Methods for producing members of specific binding pairs

ABSTRACT

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

This is a continuation of U.S. application Ser. No. 08/484,893, filedJun. 7, 1995 now U.S. Pat. No. 6,172,197, which in turn is acontinuation of U.S. application Ser. No. 07/971,857, filed Jan. 8, 1993(now U.S. Pat. No. 5,969,108), which in turn is the U.S. national phaseof PCT/GB91/01134, filed 10 Jul. 1991.

The present invention relates to methods for producing members ofspecific binding pairs. The present invention also relates to thebiological binding molecules produced by these methods.

Owing to their high specificity for a given antigen, the advent ofmonoclonal antibodies (Kohler, G. and Milstein C; 1975 Nature 256: 495)represented a significant technical break-through with importantconsequences both scientifically and commercially.

Monoclonal antibodies are traditionally made by establishing an immortalmammalian cell line which is derived from a single immunoglobulinproducing cell secreting one form of a biologically functional antibodymolecule with a particular specificity. Because the antibody-secretingmammalian cell line is immortal, the characteristics of the antibody arereproducible from batch to batch. The key properties of monoclonalantibodies are their specificity for a particular antigen and thereproducibility with which they can be manufactured.

Structurally, the simplest antibody (IgG) comprises four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulphide bonds (see FIG. 1). The light chains exist in two distinctforms called kappa (K) and lambda (λ). Each chain has a constant region(C) and a variable region (V). Each chain is organized into a series ofdomains. The light chains have two domains, corresponding to the Cregion and the other to the V region. The heavy chains have fourdomains, one corresponding to the V region and three domains (1, 2 and3) in the C region. The antibody has two arms (each arm being a Fabregion), each of which has a VL and a VH region associated with eachother. It is this pair of V regions (VL and VH) that differ from oneantibody to another (owing to amino acid sequence variations), and whichtogether are responsible for recognising the antigen and providing anantigen binding site (ABS). In even more detail, each V region is madeup from three complementarity determining regions (CDR) separated byfour framework regions (FR). The CDR's are the most variable part of thevariable regions, and they perform the critical antigen bindingfunction. The CDR regions are derived from many potential germ linesequences via a complex process involving recombination, mutation andselection.

It has been shown that the function of binding antigens can be performedby fragments of a whole antibody. Example binding fragments are (i) theFab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (iv)the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989) whichconsists of a VH domain; (v) isolated CDR regions; and (vi) F(ab′)₂fragments, a bivalent fragment comprising two Fab fragments linked by adisulphide bridge at the hinge region.

Although the two domains of the Fv fragment are coded for by separategenes, it has proved possible to make a synthetic linker that enablesthem to be made as a single protein chain (known as single chain Fv(scFv); Bird, R. E. et al., Science 242, 423-426 (1988) Huston, J. S. etal., Proc. Natl. Acad. Sci., USA 85, 5879-5883 (1988)) by recombinantmethods. These scFv fragments were assembled from genes from monoclonalsthat had been previously isolated. In this application, the applicantsdescribe a process to assemble scFv fragments from VH and VL domainsthat are not part of an antibody that has been previously isolated.

Whilst monoclonal antibodies, their fragments and derivatives have beenenormously advantageous, there are nevertheless a number of limitationsassociated with them.

Firstly, the therapeutic applications of monoclonal antibodies producedby human immortal cell lines holds great promise for the treatment of awide range of diseases (Clinical Applications of Monoclonal Antibodies.Edited by E. S. Lennox. British Medical Bulletin 1984. PublishersChurchill Livingstone). Unfortunately, immortal antibody-producing humancell lines are very difficult to establish and they give low yields ofantibody (approximately 1 μg/ml). In contrast, equivalent rodent celllines yield high amounts of antibody (approximately 100 μg/ml). However,the repeated administration of these foreign rodent proteins to humanscan lead to harmful hypersensitivity reactions. In the main therefore,these rodent-derived monoclonal antibodies have limited therapeutic use.

Secondly, a key aspect in the isolation of monoclonal antibodies is howmany different clones of antibody producing cells with differentspecificities, can be practically established and sampled compared tohow many theoretically need to be sampled in order to isolate a cellproducing antibody with the desired specificity characteristics(Milstein, C., Royal Soc. Croonian Lecture, Proc. R. Soc. London B. 239;1-16, (1990)). For example, the number of different specificitiesexpressed at any one time by lymphocytes of the murine immune system isthought to be approximately 10⁷ and this is only a small proportion ofthe potential repertoire of specificities. However, during the isolationof a typical antibody producing cell with a desired specificity, theinvestigator is only able to sample 10³ to 10⁴ individual specificities.The problem is worse in the human, where one has approximately 10¹²lymphocyte specificities, with the limitation on sampling of 10³ or 10⁴remaining.

This problem has been alleviated to some extent in laboratory animals bythe use of immunisation regimes. Thus, where one wants to producemonoclonal antibodies having a specificity against a particular epitope,an animal is immunised with an immunogen expressing that epitope. Theanimal will then mount an immune response against the immunogen andthere will be a proliferation of lymphocytes which have specificityagainst the epitope. Owing to this proliferation of lymphocytes with thedesired specificity, it becomes easier to detect them in the samplingprocedure. However, this approach is not successful in all cases, as asuitable immunogen may not be available. Furthermore, where one wants toproduce human monoclonal antibodies (eg for therapeutic administrationas previously discussed), such an approach is not practically, orethically, feasible.

In the last few years, these problems have in part, been addressed bythe application of recombinant DNA methods to the isolation andproduction of e.g. antibodies and fragments of antibodies with antigenbinding ability, in bacteria such as E. coli.

This simple substitution of immortalised cells with bacterial cells asthe ‘factory’, considerably simplifies procedures for preparing largeamounts of binding molecules. Furthermore, a recombinant productionsystem allows scope for producing tailor-made antibodies and fragmentsthereof. For example, it is possible to produce chimeric molecules withnew combinations of binding and effector functions, humanised antibodies(e.g. murine variable regions combined with human constant domains ormurine-antibody CDRs grafted onto a human FR) and novel antigen-bindingmolecules. Furthermore, the use of polymerase chain reaction (PCR)amplification (Saiki, R. K., et al., Science 239, 487-491 (1988)) toisolate antibody producing sequences from cells (e.g. hybridomas and Bcells) has great potential for speeding up the timescale under whichspecificities can be isolated. Amplified VH and VL genes are cloneddirectly into vectors for expression in bacteria or mammalian cells(Orlandi, R., et al., 1989, Proc. Natl. Acad. Sci., USA 86, 3833-3837;Ward, E. S., et al., 1989 supra; Larrick, J. W., et al., 1989, Biochem.Biophys. Res. Commun. 160, 1250-1255; Sastry, L. et al., 1989, Proc.Natl. Acad. Sci., USA., 86, 5728-5732). Soluble antibody fragmentssecreted from bacteria are then screened for binding activities.

However, like the production system based upon immortalised cells, therecombinant production system still suffers from the selection problemspreviously discussed and therefore relies on animal immunization toincrease the proportion of cells with desired specificity. Furthermore,some of these techniques can exacerbate the screening problems. Forexample, large separate H and L chain libraries have been produced fromimmunized mice and combined together in a random combinatorial mannerprior to screening (Huse, W. D. et al., 1989, Science 246, 1275-1281,WO90/14443; WO90/14424 and WO90/14430). Crucially however, theinformation held within each cell, namely the original pairing of one Lchain with one H chain, is lost. This loses some, of the advantagegained by using immunization protocols in the animal. Currently, onlylibraries derived from single VH domains (dAbs; Ward, E. S., et al.,1989, supra.) do not suffer this drawback. However, because not allantibody VH domains are capable of binding antigen, more have to bescreened. In addition, the problem of directly screening many differentspecificities in prokaryotes remains to be solved.

Thus, there is a need for a screening system which ameliorates orovercomes one or more of the above or other problems. The ideal systemwould allow the sampling of very large numbers of specificities (eg 10⁶and higher), rapid sorting at each cloning round, and rapid transfer ofthe genetic material coding for the binding molecule from one stage ofthe production process, to the next stage.

The most attractive candidates for this type of screening, would beprokaryotic organisms (because they grow quickly, are relatively simpleto manipulate and because large numbers of clones can be created) whichexpress and display at their surface a functional binding domain eg. anantibody, receptor, enzyme etc. In the UK patent GB 2137631B methods forthe co-expression in a single host cell of the variable H and L chaingenes of immunoglobulins were disclosed. However, the protein wasexpressed intracellularly and was insoluble. Further, the proteinrequired extensive processing to generate antibody fragments withbinding activity and this generated material with only a fraction of thebinding activity expected for antibody fragments at this concentration.It has already been shown that antibody fragments can be secretedthrough bacterial membranes with the appropriate signal peptide (Skerra,A. and Pluckthun, A. 1988 Science 240 1038-1040; Better, M et al 1988,Science 240 1041-1043) with a consequent increase in the bindingactivity of antibody fragments. These methods require screening ofindividual clones for binding activity in the same way as do mousemonoclonal antibodies.

It has not been shown however, how a functional binding domain eg anantibody, antibody fragment, receptor, enzyme etc can be held on thebacterial surface in a configuration which allows sampling of say itsantigen binding properties and selection for clones with desirableproperties. In large part, this is because the bacterial surface is acomplex structure, and in the gram-negative organisms there is an outerwall which further complicates the position. Further, it has not beenshown that eg an antibody domain will fold correctly when expressed as afusion with a surface protein of bacteria or bacteriophage.

Bacteriophage are attractive prokaryote related organisms for this typeof screening. In general, their surface is a relatively simplestructure, they can be grown easily in large numbers, they are amenableto the practical handling involved in many potential mass screeningprogrammes, and they carry genetic information for their own synthesiswithin a small, simple package. The difficulty has been to practicallysolve the problem of how to use bacteriophages in this manner. A GenexCorporation patent application number WO88/06630 has proposed that thebacteriophage lambda would be a suitable vehicle for the expression ofantibody molecules, but they do not provide a teaching which enables thegeneral idea to be carried out. For example WO88/06630 does notdemonstrate that any sequences: (a) have been expressed as a fusion withgene V; (b) have been expressed on the surface of lambda; and (c) havebeen expressed so that the protein retains biological activity.Furthermore there is no teaching on how to screen for suitable fusions.Also, since the lambda virions are assembled within the cell, the fusionprotein would be expressed intracellularly and would be predicted to beinactive. Bass et al., in December 1990 (after the earliest prioritydate for the present application) describe deleting part of gene III ofthe filamentous bacteriophage M13 and inserting the coding sequence forhuman growth hormone (hGH) into the N-terminal site of the gene. Thegrowth hormone displayed by M13 was shown to be functional. (Bass, S.,et al. Proteins, Structure, Function and Genetics (1990) 8: 309-314). Afunctional copy of gene III was always present in addition, when thisfusion was expressed. A Protein Engineering Corporation patentapplication WO90/02809 proposes the insertion of the coding sequence forbovine pancreatic trypsin inhibitor (BPTI) into gene VIII of M13.However, the proposal was not shown to be operative. For example, thereis no demonstration of the expression of BPTI sequences as fusions withprotein VIII and display on the surface of M13. Furthermore thisdocument teaches that when a fusion is made with gene III, it isnecessary to use a second synthetic copy of gene III, so that someunaltered gene III protein will be present. The embodiments of thepresent application do not do this. In embodiments where phagemid isrescued with M13K07 gene III deletion phage, there is no unaltered geneIII present.

WO90/02809 also teaches that phagemids that do not contain the fullgenome of M13 and require rescue by coinfection with helper phage arenot suitable for these purposes because coinfection could lead torecombination.

In all embodiments where the present applicants have used phagemids,they have used a helper phage and the only sequences derived fromfilamentous bacteriophage in the phagemids are the origin of replicationand gene III sequences.

WO90/02809 also teaches that their process needed information such asnucleotide sequence of the starting molecule and its three-dimensionedstructure. The use of a pre-existing repertoire of binding molecules toselect for a binding member, such as is disclosed herein, for exampleusing an immunoglobulin gene repertoire of animals, was not disclosed.Further, they do not discuss favouring variegation of their bindingmolecules in natural blocks of variation such as CDRs ofimmunoglobulins, in order to favour generation of improved molecules andprevent unfavourable variations. WO90/02809 also specifically excludedthe application of their process to the production of scFv molecules.

In each of the above discussed patents (WO88/06630 and WO90/02809), theprotein proposed for display is a single polypeptide chain. There is nodisclosure of a method for the display of a dimeric molecule byexpression of one monomer as a fusion with a capsid protein and theother protein in a free form.

Another disclosure published in May 1991 (after the earliest prioritydate for the present application) describes the insertion into gene VIIIof M13, the coding sequences for one of the two chains of the Fabportion of an antibody with co-expression of the other from a plasmid.The two chains were demonstrated as being expressed as a functional Fabfragment on the surface of the phage (Kang A. S. et al., (1991) Proc.Natl. Acad. Sci, USA, 88 p 4363-4366). No disclosure was made of thesite of insertion into gene VIII and the assay for pAb binding activityby ELISA used a reagent specific for antibody L chain rather than forphage. A further disclosure published in March 1991 (after the earliestpriority date for the present application) describes the insertion of afragment of the AIDS virus protein gag into the N-terminal portion ofgene III of the bacteriophage fd. The expression of the gag proteinfragment was detected by immunological methods, but it was not shownwhether or not the protein was expressed in a functional form(Tsunetsugu-Yokota Y et al. (1991) Gene 99 p 261-265).

The problem of how to use bacteriophages in this way is in fact adifficult one. The protein must be inserted into the phage in such a waythat the integrity of the phage coat is not undermined, and the proteinitself should be functional retaining its biological activity withrespect to antigen binding. Thus, where the protein of choice is anantibody, it should fold efficiently and correctly and be presented forantigen binding. Solving the problem for antibody molecules andfragments would also provide a general method for any biomolecule whichis a member of a specific binding pair e.g. receptor molecules andenzymes.

Surprisingly, the applicants have been able to construct a bacteriophagethat expresses and displays at its surface a large biologicallyfunctional binding molecule (eg antibody fragments, and enzymes andreceptors) and which remains intact and infectious. The applicants havecalled the structure which comprises a virus particle and a bindingmolecule displayed at the viral surface a ‘package’. Where the bindingmolecule is an antibody, an antibody derivative or fragment, or a domainthat is homologous to an immunoglobulin domain, the applicants call thepackage a ‘phage antibody’ (pAb). However, except where the contextdemands otherwise, where the term phage antibody is used generally, itshould also be interpreted as referring to any package comprising avirus particle and a biologically functional binding molecule displayedat the viral surface.

pAbs have a range of applications in selecting antibody genes encodingantigen binding activities. For example, pAbs could be used for thecloning and rescue of hybridomas (Orlandi, R., et al (1989) PNAS 86 p3833-3837), and in the screening of large combinatorial libraries (suchas found in Huse, W. D. et al., 1989, Science 246, 1275-1281). Inparticular, rounds of selection using pAbs may help in rescuing thehigher affinity antibodies from the latter libraries. It may bepreferable to screen small libraries derived from antigen-selected cells(Casali, P., et al., (1986) Science 234 p 476-479) to rescue theoriginal VH/VL pairs comprising the Fv region of an antibody. The use ofpAbs may also allow the construction of entirely synthetic antibodies.Furthermore, antibodies may be made which have some synthetic sequencese.g. CDRs, and some naturally derived sequences. For example, V-generepertoires could be made in vitro by combining un-rearranged V genes,with D and J segments. Libraries of pAbs could then be selected bybinding to antigen, hypermutated in vitro in the antigen-binding loopsor V domain framework regions, and subjected to further rounds ofselection and mutagenesis.

As previously discussed, separate H and L chain libraries lose theoriginal pairing between the chains. It is difficult to make and screena large enough library for a particularly advantageous combination of Hand L chains.

For example, in a mouse there are approximately 10⁷ possible H chainsand 10⁷ possible L chains. Therefore, there are 10¹⁴ possiblecombinations of H and L chains, and to test for anything like thisnumber of combinations one would have to create and screen a library ofabout 10¹⁴ clones. This has not previously been a practical possibility.

The present invention provides a number of approach s which amelioratethis problem.

In a first approach, (a random combinatorial approach, see examples 20and 21) as large a library as is practically possible is created whichexpresses as many of the 10¹⁴ potential combinations as possible.However, by virtue of the expression of the H and L chains on thesurface of the phage, it is reasonably practicable to select the desiredcombination, from all the generated combinations by affinity techniques(see later for description of selection formats).

In a second approach (called a dual combinatorial approach by thepresent applicants, see example 26), a large library is created from twosmaller libraries for selection of the desired combination. Thisameliorates the problems still further. The approach involves thecreation of: (i) a first library of say 10⁷ e.g. H chains which aredisplayed on a bacteriophage (as a fusion with the protein encoded bygene III) which is resistant to e.g. tetracycline; and (ii) a secondlibrary of say 10⁷ e.g. L chains in which the coding sequences for theselight chains are within a plasmid vector containing an origin ofreplication for a bacteriophage (a phagemid) which is resistant to e.g.ampicillin (i.e. a different antibiotic) and are expressed in theperiplasmic space of a host bacterium. The first library is then used toinfect the bacteria containing the second library to provide 10¹⁴combinations of H and L chains on the surface of the resulting phage inthe bacterial supernatant.

The advantage of this approach is that two separate libraries of eg 10⁷are created in order to produce 10¹⁴ combinations. Creating a 10⁷library is a practical possibility.

The 10¹⁴ combinations are then subjected to selection (see later fordescription of selection formats) as disclosed by the presentapplication. This selection will then produce a population of phagesdisplaying a particular combination of H and L chains having the desiredspecificity. The phages selected however, will only contain DNA encodingone partner of the paired H and L chains (deriving from either the phageor phagemid). The sample eluate containing the population is thendivided into two portions. A first portion is grown on e.g. tetracyclineplates to select those bacteriophage containing DNA encoding H chainswhich are involved in the desired antigen binding. A second portion isgrown on e.g. ampicillin plates to select those bacteriophage containingphagemid DNA encoding L chains which are involved in the desired antigenbinding. A set of colonies from individually isolated clones e.g. fromthe tetracycline plates are then used to infect specific colonies e.g.from the ampicillin plates. This results in bacteriophage expressingspecific combinations of H and L chains which can then be assayed forantigen binding.

In a third approach (called a hierarchical dual combinational approachby the present applicants), an individual colony from either the H or Lchain clone selected by growth on the antibiotic plates, is used toinfect a complete library of clones encoding the other chain (H or L).Selection is as described above. This favours isolation of the mostfavourable combination.

In a fourth approach (called a hierarchical approach by the presentapplicants, see examples 22 and 46) both chains are cloned into the samevector. However, one of the chains which is already known to havedesirable properties is kept fixed. A library of the complementary chainis inserted into the same vector. Suitable partners for the fixed chainare selected following display on the surface of bacteriophage.

In a fifth approach (see example 48), to improve the chances ofrecovering original pairs, the complexity of the combinatorial librariescan be reduced by using small B populations of B-lymphocytes selectedfor binding to a desired antigen. The cells provide e.g. mRNA or DNA,for preparing libraries of antibody genes for display on phage. Thistechnique can be used in combination with the above mentioned fourapproaches for selection of antibody specificities.

Phagemids have been mentioned above. The applicants have realised anddemonstrated that in many cases phagemids will be preferred to phage forcloning antibodies because it is easier to use them to generate morecomprehensive libraries of the immune repertoire. This is because thephagemid•DNA is approximately 100 times more efficient thanbacteriophage DNA in transforming bacteria (see example 19). Also, theuse of phagemids gives the ability to vary the number of gene IIIbinding molecule fusion proteins displayed on the surface of thebacteriophage (see example 17). For example, in a system comprising abacterial cell containing a phagemid encoding a gene III fusion proteinand infected with a helper phage, induction of expression of the geneIII fusion protein to different extents, will determine the number ofgene III fusion proteins present in the space defined between the innerand outer bacterial membranes following superinfection. This willdetermine the ratio of gene III fusion protein to native gene IIIprotein displayed by the assembled phage.

Expressing a single fusion protein per virion may aid selection ofantibody specificities on the basis of affinity by avoiding the‘avidity’ effect where a phage expressing two copies of a low affinityantibody would have the same apparent affinity as a phage expressing onecopy of a higher affinity antibody. In some cases however, it will beimportant to display all the gene III molecules derived bysuperinfection of cells containing phagemids to have fusions (e.g. forselecting low affinity binding molecules or improving sensitivity onELISA). One way to do this is to superinfect with a bacteriophage whichcontains a defective gene III. The applicants have therefore developedand used a phage which is deleted in gene III. This is completely novel.

The demonstration that a functional antigen-binding domain can bedisplayed on the surface of phage, has implications beyond theconstruction of novel antibodies. For example, if other protein domainscan be displayed at the surface of a phage, phage vectors could be usedto clone and select genes by the binding properties of the displayedprotein. Furthermore, variants of proteins, including epitope librariesbuilt into the surface of the protein, could be made and readilyselected for binding activities. In effect, other protein architecturesmight serve as “nouvelle” antibodies.

The technique provides the possibility of building antibodies from firstprinciples, taking advantage of the structural framework on which theantigen binding loops fold. In general, these loops have a limitednumber of conformations which generate a variety of binding sites byalternative loop combinations and by diverse side chains. Recentsuccesses in modelling antigen binding sites augurs well for de novodesign. In any case, a high resolution structure of the antigen isneeded. However, the approach is attractive for making e.g. catalyticantibodies, particularly for small substrates. Here side chains orbinding sites for prosthetic groups might be introduced, not only tobind selectively to the transition state of the substrate, but also toparticipate directly in bond making and breaking. The only question iswhether the antibody architecture, specialised for binding, is the beststarting point for building catalysts. Genuine enzyme architectures,such as the triose phosphate isomerase (TIM) barrel, might be moresuitable. Like antibodies, TIM enzymes also have a framework structure(a barrel of β-strands and α-helices) and loops to bind substrate. Manyenzymes with a diversity of catalytic properties are based on thisarchitecture and the loops might be manipulated independently on theframeworks for design of new catalytic and binding properties. The phageselection system as provided by the present disclosure can be used toselect for antigen binding activities and the CDR loops thus selected,used on either an antibody framework or a TIM barrel framework. Loopsplaced on a e.g. a TIM barrel framework could be further modified bymutagenesis and subjected to further selection. Thus, there is no needto select for high affinity binding activities in a single step. Thestrategy of the immune system, in which low affinity evolves to highaffinity seems more realistic and can be mimicked using this invention.

One class of molecules that could be useful in this type of applicationare receptors. For example, a specific receptor could be displayed onthe surface of the phage such that it would bind its ligand. Thereceptor could then be modified by, for example, in vitro mutagenesisand variants having higher binding affinity for the ligand selected. Theselection may be carried out according to one or more of the formatsdescribed below with reference to FIG. 2 (which refers particularly topAbs) in which the pAb antibody is replaced with a phage receptor andthe antigen with a ligand 1.

Alternatively, the phage-receptor could be used as the basis of a rapidscreening system for the binding of ligands, altered ligands, orpotential drug candidates. The advantages of this system namely ofsimple cloning, convenient expression, standard reagents and easyhandling makes the drug screening application particularly attractive.In the context of this discussion, receptor means a molecule that bindsa specific, or group of specific, ligand(s). The natural receptor couldbe expressed on the surface of a population of cells, or it could be theextracellular domain of such a molecule (whether such a form existsnaturally or not), or a soluble molecule performing a natural bindingfunction in the plasma, or within a cell or organ.

Another possibility, is the display of an enzyme molecule or active siteof an enzyme molecule on the surface of a phage (see examples 11, 12,30, 31, 32 and 36). Once the phage enzyme is expressed, it can beselected by affinity chromatography, for instance on columns derivatizedwith transition state analogues. If an enzyme with a different ormodified specificity is desired, it may be possible to mutate an enzymedisplayed as a fusion on bacteriophage and then select on a columnderivatised with an analogue selected to have a higher affinity for anenzyme with the desired modified specificity.

Although throughout this application, the applicants discuss thepossibility of screening for higher affinity variants of pAbs, theyrecognise that in some applications, for example low affinitychromatography (Ohlson, S. et al Anal. Biochem. 169, p 204-208 (1988)),it may be desirable to isolate lower affinity variants.

Examples 21 and 23 show that the present invention provides a way ofproducing antibodies with low affinities (as seen in the primary immuneresponse or in unimmunised animals). This is made possible by displayingmultiple copies of the antibody on the phage surface in association withgene III protein. Thus, pAbs allow genes for these antibodies to beisolated and if necessary, mutated to provide improved antibodies.

pAbs also allow the selection of antibodies for improved stability. Ithas been noted for many antibodies, that yield and stability areimproved when the antibodies are expressed at 30° C. rather than 37° C.If pAbs are displayed at 37° C., only those which are stable will beavailable for affinity selection. When antibodies are to be used in vivofor therapeutic or diagnostic purposes, increased stability would extendthe half-life of antibodies in circulation.

Although stability is important for all antibodies and antibody domainsselected using phage, it is particularly important for the selection ofFv fragments which are formed by the non-covalent association of VH andVL fragments. Fv fragments have a tendency to dissociate and have a muchreduced half-life in circulation compared to whole antibodies. Fvfragments are displayed on the surface of phage, by the association ofone chain expressed as a gene III protein fusion with the complementarychain expressed as a soluble fragment. If pairs of chains have a hightendency to dissociate, they will be much less likely to be selected aspAbs. Therefore, the population will be enriched for pairs which doassociate stably. Although dissociation is less of a problem with Fabfragments, selection would also occur for Fab fragments which associatestably. pAbs allow selection for stability to protease attack, onlythose pAbs that are not cleaved by proteases will be capable of bindingtheir ligand and therefore populations of phage will be enriched forthose displaying stable antibody domains.

The technique of displaying binding molecules on the phage surface canalso be used as a primary cloning system. For example, a cDNA librarycan be constructed and inserted into the bacteriophage and this phagelibrary screened for the ability to bind a ligand. The ligand/bindingmolecule combination could include any pair of molecules with an abilityto specifically bind to one another e.g. receptor/ligand,enzyme/substrate (or analogue), nucleic acid binding protein/nucleicacid etc. If one member of the complementary pair is available, this maybe a preferred way of isolating a clone for the other member of thepair.

It will often be necessary to increase the diversity of a population ofgenes cloned for the display of their proteins on phage or to mutate anindividual nucleotide sequence. Although in vitro or in vivo mutagenesistechniques could be used for either purpose, a particularly suitablemethod would be to use mutator strains. A mutator strain is a strainwhich contains a genetic defect which causes DNA replicated within it tobe mutated with respect to its parent DNA. Hence if a population ofgenes as gene III fusions is introduced into these strains it will befurther diversified and can then be transferred to a non-mutator strain,if desired, for display and selection. Example 38 covers the use ofmutator strains with phage antibodies (an example of in vitromutagenesis and selection of phage antibodies is given in example 45).

Targeted Gene Transfer

A useful and novel set of applications makes use of the binding proteinon the phage to target the phage genome to a particular cell or group ofcells. For example, a pAb specific for a cell surface molecule could beused to bind to the target cell via the surface molecule. The phagecould then be internalised, either through the action of the receptoritself or as the result of another event (e.g. an electrical dischargesuch as in the technique of electroporation). The phage genome wouldthen be expressed if the relevant control signals (for transcription andtranslation and possibly replication) were present. This would beparticularly useful if the phage genome contained a sequence whoseexpression was desired in the target cell (along with the appropriateexpression control sequences). A useful sequence might confer antibioticresistance to the recipient cell or label the cell by the expression ofits product (e.g. if the sequence expressed a detectable gene productsuch as a luciferase, see White, M, et al, Techniques 2(4), p 194-201(1990)), or confer a particular property on the target cell (e.g. if thetarget cell was a tumour cell and the new sequence directed theexpression of a tumour suppressing gene), or express an antisenseconstruct designed to turn off a gene or set of genes in the targetcell, or a gene or gene product designed to be toxic to the target cell.Alternatively, the sequence whose expression is desired in the targetcell can be encoded on a phagemid. The phagemid DNA may then beincorporated into a phage displaying an antibody specific for a cellsurface receptor. For example, incorporation may be by superinfection ofbacteria containing the phagemid, with a helper phage whose genomeencodes the antibody fragment specific for the target cell. The packageis then used to direct the phagemid to the target cell.

This technique of “targeted gene transfer” has a number of uses inresearch and also in therapy and diagnostics. For example, gene therapyoften aims to target the replacement gene to a specific cell type thatis deficient in its activity. Targeting pAbs provide a means ofachieving this.

In diagnostics, phage specific for particular bacteria or groups ofbacteria have been used to target marker genes, e.g. luciferase, to thebacterial host (sec, for example, Ulitzer, S., and Kuhn, J., EPA85303913.9). If the host range of the phage is appropriate, only thosebacteria that are being tested for, will be infected by the phage,express the luciferase gene and be detected by the light they emit. Thissystem has been used to detect the presence of Salmonella. One majorproblem with this approach is the initial isolation of a bacteriophagewith the correct host range and then the cloning of a luciferase genecassette into that phage, such that it is functional. The pAb systemallows the luciferase cassette to be cloned into a well characterisedsystem (filamentous phage) and allows simple selection of an appropriatehost range, by modifying the antibody (or other binding molecule)specificity that the pAb encodes.

The present applicants have also been able to develop novel selectionsystems and assay formats which depend on the unique properties of thesereplicable genetic display packages e.g. pAbs.

TERMINOLOGY

Much of the terminology discussed in this section has been mentioned inthe text where appropriate.

Specific Binding Pair

This describes a pair of molecules (each being a member of a specificbinding pair) which are naturally derived or synthetically produced. Oneof the pair of molecules, has an area on its surface, or a cavity whichspecifically binds to, and is therefore defined as complementary with aparticular spatial and polar organisation of the other molecule, so thatthe pair have the property of binding specifically to each other.Examples of types of specific binding pairs are antigen-antibody,biotin-avidin, hormone-hormone receptor, receptor-ligand,enzyme-substrate, IgG-protein A.

Multimeric Member

This describes a first polypeptide which will associate with at least asecond polypeptide, when the polypeptides are expressed in free formand/or on the surface of a substrate. The substrate may be provided by abacteriophage. Where there are two associated polypeptides, theassociated polypeptide complex is a dimer, where there are three, atrimer etc. The dimer, trimer, multimer etc or the multimeric member maycomprise a member of a specific binding pair.

Example multimeric members are heavy domains based on an immunoglobulinmolecule, light domains based on an immunoglobulin molecule, T-cellreceptor subunits.

Replicable Genetic Display Package (Rgdp)

This describes a biological particle which has genetic informationproviding the particle with the ability to replicate. The particle candisplay on its surface at least part of a polypeptide. The polypeptidecan be encoded by genetic information native to the particle and/orartificially placed into the particle or an ancestor of it. Thedisplayed polypeptide may be any member of a specific binding pair eg.heavy or light chain domains based on an immunoglobulin molecule, anenzyme or a receptor etc.

The particle may be a virus eg. a bacteriophage such as fd or M13.

Package

This describes a replicable genetic display package in which theparticle is displaying a member of a specific binding pair at itssurface. The package may be a bacteriophage which displays an antigenbinding domain at its surface. This type of package has been called aphage antibody (pAb).

Antibody

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any protein having abinding domain which is homologous to an immunoglobulin binding domain.These proteins can be derived from natural sources, or partly or whollysynthetically produced.

Example antibodies are the immunoglobulin isotypes and the Fab, F(ab¹)₂,scFv, Fv, dAb, Fd fragments.

Immunoglobulin Superfamily

This describes a family of polypeptides, the members of which have atleast one domain with a structure related to that of the variable orconstant domain of immunoglobulin molecules. The domain contains twoβ-sheets and usually a conserved disulphide bond (see A. F. Williams andA. N. Barclay 1988 Ann. Rev Immunol. 6 381-405).

Example members of an immunoglobulin superfamily are CD4, plateletderived growth factor receptor (PDGFR), intercellular adhesion molecule.(ICAM). Except where the context otherwise dictates, reference toimmunoglobulins and immunoglobulin homologs in this application includesmembers of the immunoglobulin superfamily and homologs thereof.

Homologs

This term indicates polypeptides having the same or conserved residuesat a corresponding position in their primary, secondary or tertiarystructure. The term also extends to two or more nucleotide sequencesencoding the homologous polypeptides.

Example homologous peptides are the immunoglobulin isotypes.

Functional

In relation to a sbp member displayed on the surface of a rgdp, meansthat the sbp member is presented in a folded form in which its specificbinding domain for its complementary sbp member is the same or closelyanalogous to its native configuration, whereby it exhibits similarspecificity with respect to the complementary sbp member. In thisrespect, it differs from the peptides of Smith et al, supra, which donot have a definite folded configuration and can assume a variety ofconfigurations determined by the complementary members with which theymay be contacted.

Genetically Diverse Population

In connection with sbp members or polypeptide components thereof, thisis referring not only to diversity that can exist in the naturalpopulation of cells or organisms, but also diversity that can be createdby artificial mutation in vitro or in vivo.

Mutation in vitro may for example, involve random mutagenesis usingoligonucleotides having random mutations of the sequence desired to bevaried. In vivo mutagenesis may for example, use mutator strains of hostmicroorganisms to harbour the DNA (see Example 38 below).

Domain

A domain is a part of a protein that is folded within itself andindependently of other parts of the same protein and independently of acomplementary binding member.

Folded Unit

This is a specific combination of an α-helix and/or β-strand and/orβ-turn structure. Domains and folded units contain structures that bringtogether amino acids that are not adjacent in the primary structure.

Free Form

This describes the state of a polypeptide which is not displayed by areplicable genetic display package.

Conditionally Defective

This describes a gene which does not express a particular polypeptideunder one set of conditions, but expresses it under another set ofconditions. An example, is a gene containing an amber mutation expressedin non-suppressing or suppressing hosts respectively.

Alternatively, a gene may express a protein which is defective under oneset of conditions, but not under another set. An example is a gene witha temperature sensitive mutation.

Suppressible Translational Stop Codon

This describes a codon which allows the translation of nucleotidesequences downstream of the codon under one set of conditions, but underanother set of conditions translation ends at the codon. Example ofsuppressible translational stop codons are the amber, ochre and opalcodons.

Mutator Strain

This is a host cell which has a genetic defect which causes DNAreplicated within it to be mutated with respect to its parent DNA.Example mutator strains are NR9046mutD5 and NR9046 mut T1 (see Example38).

Helper Phage

This is a phage which is used to infect cells containing a defectivephage genome and which functions to complement the defect. The defectivephage genome can be a phagemid or a phage with some function encodinggene sequences removed. Examples of helper phages are M13KO7, M13K07gene III no. 3; and phage displaying or encoding a binding moleculefused to a capsid protein.

Vector

This is a DNA molecule, capable of replication in a host organism, intowhich a gene is inserted to construct a recombinant DNA molecule.

Phage Vector

This is a vector derived by modification of a phage genome, containingan origin of replication for a bacteriophage, but not one for a plasmid.

Phagemid Vector

This is a vector derived by modification of a plasmid genome, containingan origin of replication for a bacteriophage as well as the plasmidorigin of replication.

Secreted

This describes a rgdp or molecule that associates with the member of asbp displayed on the rgdp, in which the sbp member and/or the molecule,have been folded and the package assembled externally to the cellularcytosol.

Repertoire of Rearranged Immunoglobulin Genes

A collection of naturally occurring nucleotides eg DNA sequences whichencoded expressed immunoglobulin genes in an animal. The sequences aregenerated by the in vivo rearrangement of eg V, D and J segments for Hchains and eg the V and J segments for L chains. Alternatively thesequences may be generated from a cell line immunised in vitro and inwhich the rearrangement in response to immunisation occursintracellularly.

Library

A collection of nucleotide eg DNA, sequences within clones.

Repertoire of Artificially Rearranged Immunoglobulin Genes

A collection of nucleotide eg DNA, sequences derived wholly or partlyfrom a source other than the rearranged immunoglobulin sequences from ananimal. This may include for example, DNA sequences encoding VH domainsby combining unrearranged V segments with D and J segments and DNAsequences encoding VL domains by combining V and J segments.

Part or all of the DNA sequences may be derived by oligonucleotidesynthesis.

Secretory Leader Peptide

This is a sequence of amino acids joined to the N-terminal end of apolypeptide and which directs movement of the polypeptide out of thecytosol.

Eluant

This is a solution used to breakdown the linkage between two molecules.The linkage can be a non-covalent or covalent bond(s). The two moleculescan be members of a sbp.

Derivative

This is a substance which derived from a polypeptide which is encoded bythe DNA within a selected rgdp. The derivative polypeptide may differfrom the encoded polypeptide by the addition, deletion, substitution orinsertion of amino acids, or by the linkage of other molecules to theencoded polypeptide. These changes may be made at the nucleotide orprotein level. For example the encoded polypeptide may be a Fab fragmentwhich is then linked to an Fc tail from another source. Alternativelymarkers such as enzymes, fluoresceins etc may be linked to eg Fab, scFvfragments.

SUMMARY OF THE INVENTION

The present invention provides a method for producing a replicablegenetic display package or population such rgdps of which methodcomprises the steps of:

-   a) inserting a nucleotide sequence encoding a member of a specific    binding pair eg. a binding molecule within a viral genome;-   b) culturing the virus containing said nucleotide sequence, so that    said binding molecule is expressed and displayed by the virus at its    surface.

The present invention also provides a method for selecting a rgdpspecific for a particular epitope which comprises producing a populationof such rgdps as described above and the additional step of selectingfor said binding molecule by contacting the population with said epitopeso that individual rgdps with the desired specificity may bind to saidepitope. The method may comprise one or more of the additional steps of:(i) separating any bound rgdps from the epitope; (ii) recovering anyseparated rgdps and (iii) using the inserted nucleotide sequences fromany separated rgdps in a recombinant system to produce the bindingmolecule separate from virus. The selection step may isolate thenucleotide sequence encoding the binding molecule of desiredspecificity, by virtue of said binding molecule being expressed inassociation with the surface of the virus in which said encoding nucleicacid is contained.

The present invention also provides a method of producing a multimericmember of a specific binding pair (sbp), which method comprises:

expressing in a recombinant host organism a first polypeptide chain ofsaid sbp member or a genetically diverse population of said sbp memberfused to a component of a secreted replicable genetic display package(rgdp) which thereby displays said polypeptide at the surface of thepackage, and expressing in a recombinant host organism a secondpolypeptide chain of said multimer and causing or allowing thepolypeptide chains come together to form said multimer as part of saidrgdp at least one of said polypeptide chains being expressed fromnucleic acid that is capable of being packaged using said componenttherefor, whereby the genetic material of each said rgdp encodes a saidpolypeptide chain. Both said chains may be expressed in the same hostorganism.

The first and second chains of said multimer may be expressed asseparate chains from a single vector containing their respective nucleicacid.

At least one of said polypeptide chains may be expressed from a phagevector.

At least one of said polypeptide chains may be expressed from a phagemidvector, the method including using a helper phage, or a plasmidexpressing complementing phage genes, to help package said phagemidgenome, and said component of the rgdp is a capsid protein therefor. Thecapsid protein may be absent, defective or conditionally defective inthe helper phage.

The method may comprise introducing a vector capable of expressing saidfirst polypeptide chain, into a host organism which expresses saidsecond polypeptide chain in free form, or introducing a vector capableof expressing said second polypeptide in free form into a host organismwhich expresses said first polypeptide chain.

Each of the polypeptide chain may be expressed from nucleic acid whichis capable of being packaged as a rgdp using said component fusionproduct, whereby encoding nucleic acid for both said polypeptide chainsare packaged in respective rgdps.

The nucleic acid encoding at least one of said first and secondpolypeptide chains may be obtained from a library of nucleic acidincluding nucleic acid encoding said chain or a population of variantsof said chain. Both the first and second polypeptide chains may beobtained from respective said libraries of nucleic acid.

The present invention also provides a method of producing a member of aspecific binding pair (sbp), from a nucleic acid library includingnucleic acid encoding said sbp member or a genetically diversepopulation of said type of sbp members, which method comprises:

-   -   expressing in recombinant host cells polypeptides encoded by        said library nucleic acid fused to a component of a secreted        replicable genetic display package (rgdp) or in free form for        association with a polypeptide component of said sbp member        which is expressed as a fusion to said rgdp component so that        the rgdp displays said sbp member in functional form at the        surface of the package, said library nucleic acid being        contained within the host cells in a form that is capable of        being packaged using said rgdp component, whereby the genetic        material of an rgdp displaying an sbp member contains nucleic        acid encoding said sbp member or a polypeptide component        thereof.

The nucleotide sequences for the libraries may be derived from eg animalspleen cells or peripheral blood lymphocytes. Alternatively thenucleotide sequence may be derived by the in vitro mutagenesis of anexisting antibody coding sequence.

The present invention also provides a method of producing a member of aspecific binding pair (sbp), which method comprises:

-   -   expressing in recombinant host cells nucleic acid encoding said        sbp member or a genetically diverse population of said type of        sbp member wherein the or each said sbp member or a polypeptide        component thereof is expressed as a fusion with a component of a        secreted replicable genetic display package (rgdp) which        displays said sbp member at the surface of the package, nucleic        acid encoding said sbp member or a polypeptide component thereof        being contained within the host cell in a form that is capable        of being packaged using said rgdp component whereby the genetic        material of the rgdp displaying said sbp member encodes said sbp        member or a polypeptide component thereof, said host organism        being a mutator strain which introduces genetic diversity into        the sbp member to produce said mixed population.

The present invention also provides a method of producing a member of aspecific binding pair (sbp), which method comprises:

-   -   expressing in recombinant host cells nucleic acid encoding said        sbp member or a genetically diverse population of said type of        sbp member wherein the or each said sbp member or a polypeptide        component thereof is expressed as a fusion with a component of a        secreted replicable genetic display package (rgdp) which        displays said sbp member in functional form at the surface of        the package, nucleic acid encoding said sbp member or a        polypeptide component thereof being contained within the host        cell in a form that is capable of being packaged using said rgdp        component whereby the genetic material of the rgdp displaying an        sbp member encodes said sbp member or a polypeptide component        thereof, said fusions being with bacteriophage capsid protein        and the rgdps being formed with said fusions in the absence of        said capsid expressed in wild-type form.

The present invention also provides a method of producing a member of aspecific binding pair (sbp) which method comprises:

-   -   expressing in recombinant host cells nucleic acid encoding said        sbp member or a genetically diverse population of said type of        sbp member or a polypeptide component thereof fused to a        component of a secreted replicable genetic display package        (rgdp) which displays said sbp member in functional form at the        surface of the package, nucleic acid encoding said sbp member or        a polypeptide component thereof being contained within the host        cell in a form that is capable of being packaged using said rgdp        component whereby the genetic material of the rgdp displaying an        sbp member or a polypeptide component thereof encodes said sbp        member or a polypeptide component thereof, said sbp member or        polypeptide component thereof being expressed from a phagemid as        a capsid fusion, and a helper phage, or a plasmid expressing        complementing phage genes, is used along with said capsid        fusions to package the phagemid nucleic acid.

The library or genetically diverse population may be obtained from:

-   -   (i) the repertoire of rearranged immunoglobulin genes of an        animal immunised with complementary sbp member,    -   (ii) the repertoire of rearranged immunoglobulin genes of an        animal not immunised with complementary sbp member,    -   (iii) a repertoire of artificially rearranged immunoglobulin        gene or genes    -   (iv) a repertoire of immunoglobulin homolog gene or genes; or    -   (v) a mixture of any of (i), (ii), (iii) and (iv).

The capsid protein may be absent, defective or conditionally defectivein the helper phage.

The host cell may be a mutator strain which introduces genetic diversityinto the sbp member nucleic acid.

The sbp member may comprise a domain which is, or is homologous to, animmunoglobulin domain.

The rgdp may be a bacteriophage, the host a bacterium, and saidcomponent of the rgdp a capsid protein for the bacteriophage. The phagemay be a filamentous phage. The phage may be selected from the class Iphages fd, M13, f1, If1, lke, ZJ/Z, Ff and the class II phages Xf, Pf1and Pf3. The phage may be fd or a derivative of fd. The derivative maybe tetracycline resistant. The said sbp member or polypeptide chainthereof may be expressed as a fusion with the gene III capsid protein ofphage fd or its counterpart in another filamentous phage. The sbp memberor polypeptide chain thereof may be inserted in the N-terminal region ofthe mature capsid protein downstream of a secretory leader peptide. Thesequence may be inserted after amino acid+1 of the mature protein. Thesite for insertion may be flanked by short sequences corresponding tosequences which occur at each end of the nucleic acid to be inserted.For example where 4 the protein domain is an immunoglobulin domain, theinsertion site in the phage may be flanked by nucleotide sequences whichcode for the first five amino acids and the last five amino acids of theIg domain. Such flanking nucleotide sequences are shown in FIG. 4(2) Band C, wherein the site-flanking nucleotide sequences encode amino acidsequences QVQLQ (SEQ ID NO:1) and VTVSS (SEQ ID NO:2) which occur ateither end of the VH domain, or QVQLQ (SEQ ID NO:1) and LEIKR (SEQ IDNO:3) which occur at either end of the Fv (combined VH+VL) domain. Eachof these sequences flanking the insertion site may include a suitablecleavage site, as shown in FIG. 4( i) and FIG. 4( ii).

Alternatively, the flanking nucleotide sequences shown in FIG. 4(2)B andC as described above, may be used to flank the insertion site for anynucleic acid to be inserted, whether or not that nucleic acid codes animmunoglobulin.

The host may be E. coli.

Nucleic acid encoding an sbp member polypeptide may be linked downstreamto a viral capsid protein through a suppressible translational stopcodon.

As previously mentioned, the present invention also provides novelselection systems and assay formats. In these systems and formats, thegene sequence encoding the binding molecule (eg. the antibody) ofdesired specificity is separated from a general population of rgdpshaving a range of specifies, by the fact of its binding to a specifictarget (eg the antigen or epitope). Thus the rgdps formed by saidexpression may be selected or screened to provide an individual sbpmember or a selected mixed population of said sbp members associated intheir respective rgdps with nucleic acid encoding said sbp member or apolypeptide chain thereof. The rgdps may be selected by affinity with amember complementary to said sbp member.

Any rgdps bound to said second member may be recovered by washing withan eluant. The washing conditions may be varied in order to obtain rgdpswith different binding affinities for said epitope. Alternatively, toobtain eg high affinity rgdps, the complementary member (eg an epitope)may be presented to the population of rgdps (eg pAbs) already bound to abinding member in which case pAbs with a higher affinity for the epitopewill displace the already bound binding member. Thus the eluant maycontain a molecule which competes with said rgdp for binding to thecomplementary sbp member. The rgdp may be applied to said complementarysbp member in the presence of a molecule which competes with saidpackage for binding to said complementary sbp member. Nucleic acidderived from a selected or screened rgdp may be used to express said sbpmember or a fragment or derivative thereof in a recombinant hostorganism. Nucleic acid from one or more rgdps may be taken and used toprovide encoding nucleic acid in a further said method to obtain anindividual sbp member or a mixed population of sbp members, or encodingnucleic acid therefor. The expression end product may be modified toproduce a derivative thereof.

The expression end product or derivative thereof may be used to preparea therapeutic or prophylactic medicament or a diagnostic product.

The present invention also provides recombinant host cells harbouring alibrary of nucleic acid fragments comprising fragments encoding agenetically diverse population of a type of member of a specific bindingpair (sbp), each sbp member or a polypeptide component thereof beingexpressed as a fusion with a component of a secretable replicablegenetic display package (rgdp), so that said sbp members are displayedon the surface of the rgdps in functional form and the genetic materialof the rgdps encode the associated sbp member or a polypeptide componentthereof. The type of sbp members may be immunoglobulins orimmunoglobulin homologs, a first polypeptide chain of which is expressedas a said fusion with a component of the rgdp and a second polypeptidechain of which is expressed in free form and associates with the fusedfirst polypeptide chain in the rgdp.

The present invention also provides a helper phage whose genome lacksnucleic acid encoding one of its capsid proteins, or whose encodingnucleic acid therefor is conditionally defective, or which encodes saidcapsid protein in defective or conditionally defective form.

The present invention also provides a bacterial host cell containing afilamentous phage genome defective for a capsid protein thereof andwherein the host cell is capable of expressing capsid proteincomplementing said defect such that infectious phage particles can beobtained therefrom. The complementing capsid protein may be expressed insaid host from another vector contained therein. The defective capsidprotein may be gene III of phage fd or its counterpart in anotherfilamentous phage.

The present invention also provides recombinant E. coli TG1 M13KO7 gIIINo. 3 (NCTC 12478).

The present invention also provides a phage antibody having the form ofa replicable genetic display package displaying on its surface infunctional form a member of a specific binding pair or a specificbinding domain thereof.

In the above methods, the binding molecule may be an antibody, or adomain that is homologous to an immunoglobulin. The antibody and/ordomain may be either naturally derived or synthetic or a combination ofboth. The domain may be a Fab, scFv, Fv dAb or Fd molecule.Alternatively, the binding molecule may be an enzyme or receptor orfragment, derivative or analogue of any such enzyme or receptor.Alternatively, the binding molecule may be a member of an immunoglobulinsuperfamily and which has a structural form based on an immunoglobulinmolecule.

The present invention also provides rgdps as defined above and membersof specific binding pairs eg. binding molecules such as antibodies,enzymes, receptors, fragments and derivatives thereof, obtainable by useof any of the above defined methods. The derivatives may comprisemembers of the specific binding pairs fused to another molecule such asan enzyme or a Fc tail.

The invention also includes kits for carrying out the methods hereof.The kits will include the necessary vectors. One such vector willtypically have an origin of replication for single strandedbacteriophage and either contain the sbp member nucleic acid or have arestriction site for its insertion in the 5′ end region of the maturecoding sequence of a phage capsid protein, and with a secretory leadercoding sequence upstream of said site which directs a fusion of thecapsid protein exogenous polypeptide to the periplasmic space.

The restriction sites in the vectors are preferably those of enzymeswhich cut only rarely in protein coding sequences.

The kit preferably includes a phagemid vector which may have the abovecharacteristics, or may contain, or have a site for insertion, of sbpmember nucleic acid for expression of the encoded polypeptide in freeform.

The kits will also contain ancillary components required for carryingout the method, the nature of such components depending of course on theparticular method employed.

Useful ancillary components may comprise helper phage, PCR primers, andbuffers and enzymes of various kinds.

PCR primers and associated reagents for use where the sbp members areantibodies may have the following characteristics:

-   (i) primers having homology to the 5′ end of the sense or anti-sense    strand of sequences encoding domains of antibodies; and-   (ii) primers including tag sequences 5′ to these homologous    sequences which incorporate restriction sites to allow insertion    into vectors; together with sequences to allow assembly of amplified    VH and VL regions to enable expression as Fv, scFv or Fab fragments.

Buffers and enzymes are typically used to enable preparation ofnucleotide sequences encoding Fv, scFv or Fab fragments derived fromrearranged or unrearranged immunoglobulin genes according to thestrategies described herein.

The applicants have chosen the filamentous F-specific bacteriophages asan example of the type of phage which could provide a vehicle for thedisplay of binding molecules e.g. antibodies and antibody fragments andderivatives thereof, on their surface and facilitate subsequentselection and manipulation.

The F-specific phages (e.g. fl, fd and M13) have evolved a method ofpropagation which does not kill the host cell and they are used commonlyas vehicles for recombinant DNA (Kornberg, A., DNA Replication, W.H.Freeman and Co., San Francisco, 1980). The single stranded DNA genome(approximately 6.4 Kb) of fd is extruded through the bacterial membranewhere it sequesters capsid sub-units, to produce mature virions. Thesevirions are 6 nm in diameter, 1 μm in length and each containapproximately 2,800 molecules of the major coat protein encoded by viralgene VIII and four molecules of the adsorption molecule gene III protein(gap) the latter is located at one end of the virion. The structure hasbeen reviewed by Webster et al., 1978 in The Single Stranded DNA Phages,557-569, Cold Spring Harbor Laboratory Press. The gene III product isinvolved in the binding of the phage to the bacterial F-pilus.

Although these phages do not kill their host during normal replication,disruption of some of their genes can lead to cell death (Kornberg, A.,1980 supra.) This places some restraint on their use. The applicantshave recognized that gene III of phage fd is an attractive possibilityfor the insertion of biologically active foreign sequences. There arehowever, other candidate sites including for example gene VIII and geneVI.

The protein itself is only a minor component of the phage coat anddisruption of the gene does not lead to cell death (Smith, G. 1988,Virology 167: 156-165). Furthermore, it is possible to insert someforeign sequences (with no biological function) into various positionswithin this gene (Smith, G. 1985 Science 228: 1315-1317, Parmley, S. F.and Smith, G. P. Gene: 73 (1988) p. 305-318, and de la Cruz, V. F., etal., 1988, J. Biol. Chem., 263: 4318-4322). Smith et al described thedisplay of peptides on the outer surface of phage but they did notdescribe the display of protein domains. Peptides can adopt a range ofstructures which can be different when in free solution, than when boundto, for example, an antibody, or when forming part of a protein(Stanfield, R. I. et al., (1990) Science 248, p 712-719). Proteins ingeneral have a well defined tertiary structure and perform theirbiological function only when adopting this structure. For example, thestructure of the antibody D1.3 has been solved in the free form and whenbound to antigen (Bhat, T. N. et al., (1990) Nature 347, p 483-485). Thegross structure of the protein is identical in each instance with onlyminor variations around the binding site for the antigen. Other proteinshave more substantial conformation changes on binding of ligand, forinstance the enzymes hexokinase and pyruvate dehydrogenase during theircatalytic cycle, but they still retain their overall pattern of folding.This structural integrity is not confined to whole proteins, but isexhibited by protein domains. This leads to the concept of a folded unitwhich is part of a protein, often a domain, which has a well definedprimary, secondary and tertiary structure and which retains the sameoverall folding pattern whether binding to a binding partner or not. Theonly gene sequence that Smith et al., described that was of sufficientsize to encode a domain (a minimum of perhaps 50 amino acids) was a 335bp fragment of a β-galactosidase corresponding to nucleotides 861-1195in the β-galactosidase gene sequence (Parmley, S.+Smith, G. P. 1988supra. This would encode 112 amino acids of a much larger 380 amino aciddomain. Therefore, prior to the present application, no substantiallycomplete domain or folded unit had been displayed on phage. In thesecases, although the infectivity of the virion was disrupted, theinserted sequences could be detected on the phage surface by use of e.g.antibodies.

The protein encoded by gene III has several domains (Pratt, D., et al.,1969 Virology 39:42-53, Grant, R. A., et al., 1981, J. Biol. Chem. 256:539-546 and Armstrong, J., et al., FEBS Lett. 135: 167-172 1981)including: (i) a signal sequence that directs the protein to the cellmembrane and which is then cleaved off; (ii) a domain that anchors themature protein into the bacterial cell membrane (and also the phagecoat); and (iii) a domain that specifically binds to the phage receptor,the F-pilus of the host bacterium. Short sequences derived from proteinmolecules have been inserted into two places within the mature molecule(Smith, G., 1985 supra., and Pariley, S. F. and Smith G. P., 1988supra.). Namely, into an inter-domain region and also between aminoacids 2 and 3 at the N-terminus. The insertion sites at the N-terminuswere more successful in maintaining the structural integrity of the geneIII protein and displaying the peptides on the surface of the phage. Byuse of antisera specific for the peptides, the peptides inserted intothis position were shown to be on the surface of the phage. Theseauthors were also able to purify the phage, using this property.However, the peptides expressed by the phage, did not possess measurablebiological functions of their own.

Retaining the biological function of a molecule when it is expressed ina radically different context to its natural state is difficult. Thedemands on the structure of the molecule are heavy. In contrast,retaining the ability to be bound by specific antisera is a passiveprocess which imposes far less rigorous demands on the structure of themolecule. For example, it is the rule rather than the exception thatpolyclonal antisera will recognise totally denatured, and biologicallyinactive, proteins on Western blots (see for example, Harlow, E. andLane, D., Antibodies, a Laboratory Manual, Cold Spring Harbor LaboratoryPress 1988). Therefore, the insertion of peptides into a region thatallows their structure to be probed with antisera teaches only that theregion allows the inserted sequences to be exposed and does not teachthat the region is suitable for the insertion of large sequences withdemanding structural constraints for the display of a molecule with abiological or binding function. In particular, it does not teach thatdomains or folded units of proteins can be displayed from sequencesinserted in this region.

This experience with Western blots is a graphic practical demonstrationwhich shows that retaining the ability to be bound by specific antiseraimposes far less rigorous demands on the structure of a polypeptide,than does folding for the retention of a biological function.

Studies have been carried out, in which E. coli have been manipulated toexpress the protein B-adrenergic receptor as a fusion with the outermembrane protein lamB. The β-adrenergic receptor was expressed in afunctional form as determined by the presence of binding activity.However, when an equivalent antibody fusion was made with lamB, theantibody fusion was toxic to the host cell.

The applicants have investigated the possibility of inserting the genecoding sequence for biologically active antibody fragments into the geneIII region of fd to express a large fusion protein. As is apparent fromthe previous discussion, this approach makes onerous demands on thefunctionality of the fusion protein. The insertion is large, encodingantibody fragments of at least 100-200 amino acids; the antibody deriveddomain must fold efficiently and correctly to display antigen-binding;and most of the functions of gene III must be retained. The applicantsapproach to the construction of the fusion molecule was designed tominimise the risk of disrupting these functions. In an embodiment of theinvention, the initial vector used was fd-tet (Zacher, A. N., et al.,1980, Gene 9, 127-140) a tetracycline resistant version of fdbacteriophage that can be propagated as a plasmid that conferstetracycline resistance to the infected E. coli host. The applicantschose to insert after the signal sequence of the fd gene III protein forseveral reasons. In particular, the applicants chose to insert afteramino acid 1 of the mature protein to retain the context for the signalpeptidase cleavage.

To retain the structure and function of gene III itself, the majority ofthe original amino acids are synthesized after the insertedimmunoglobulin sequences. The inserted immunoglobulin sequences weredesigned to include residues from the switch region that links VH-VL toCH1-CL (Lesk, A., and Chothia, C., Nature 335, 188-190, 1988).

Surprisingly, by manipulating gene III of bacteriophage fd, the presentapplicants have been able to construct a bacteriophage that displays onits surface large biologically functional antibody, enzyme, and receptormolecules whilst remaining intact and infectious. Furthermore, thephages bearing antibodies of desired specificity, can be selected from abackground of phages not showing this specificity.

The sequences coding for a population of antibody molecules and forinsertion into the vector to give expression of antibody bindingfunctions on the phage surface can be derived from a variety of sources.For example, immunised or non-immunised rodents or humans, and fromorgans such as spleen and peripheral blood lymphocytes. The codingsequences are derived from these sources by techniques familiar to thoseskilled in the art (Orlandi, R., et al., 1989 supra; Larrick, J. W., etal., 1989 supra; Chiang, Y. L., et al., 1989 Bio Techniques 7, p.360-366; Ward, E. S, et al., 1989 supra; Sastry, L., et al., 1989supra.) or by novel linkage strategies described in examples 14, 33, 40and 42. Novel strategies are described in examples 7, 25, 33, 39 and 40for displaying dimeric molecules eg Fab and Fv fragments on the surfaceof a phage. Each individual pAb in the resulting library of pAbs willexpress antibodies or antibody derived fragments that are monoclonalwith respect to their antigen-binding characteristics.

The disclosure made by the present applicants is important and providesa significant breakthrough in the technology relating to the productionof biological binding molecules, their fragments and derivatives by theuse of recombinant methods.

In standard recombinant techniques for the production of antibodies, anexpression vector containing sequences coding for the antibodypolypeptide chains is used to transform e.g. E. coli. The antibodypolypeptides are expressed and detected by use of standard screeningsystems. When the screen detects an antibody polypeptide of the desiredspecificity, one has to return to the particular transformed E. coliexpressing the desired antibody polypeptide. Furthermore, the vectorcontaining the coding sequence for the desired antibody polypeptide thenhas to be isolated for use from E. coli in further processing steps.

In the present invention however, the desired antibody polypeptide whenexpressed, is already packaged with its gene coding sequence. This meansthat when the an antibody polypeptide of desired specificity isselected, there is no need to return to the original culture forisolation of that sequence. Furthermore, in previous methods in standardrecombinant techniques, each clone expressing antibody needs to bescreened individually. The present application provides for theselection of clones expressing antibodies with desired properties andthus only requires screening of clones from an enriched pool.

Because a rgdp (eg a pAb) is a novel structure that displays a member ofa specific binding pair (eg. an antibody of monoclonal antigen-bindingspecificity) at the surface of a relatively simple replicable structurealso containing the genetic information encoding the member, rgdps egpAbs, that bind to the complementary member of the specific binding pair(eg antigen) can be recovered very efficiently by either eluting off thecomplementary member using for example diethylamine, high salt etc andinfecting suitable bacteria, or by denaturing the structure, andspecifically amplifying the sequences encoding the member using PCR.That is, there is no necessity to refer back to the original bacterialclone that gave rise to the pAb.

For some purposes, for example immunoprecipitation and some diagnostictests, it is advantageous to use polyclonal antibodies or antibodyfragments. The present invention allows this to be achieved by eitherselection of an enriched pool of pAbs with desired properties or bymixing individually isolated clones with desired properties. Theantibodies or antibody fragments may then be expressed in soluble formif desired. Such a selected polyclonal pAb population can be grown fromstocks of phage, bacteria containing phagemids or bacteria expressingsoluble fragments derived from the selected polyclonal population. Thusa reagent equivalent to a polyclonal antiserum is created which can bereplicated and routinely manufactured in culture without use of animals.

Selection Formats and Affinity Maturation

Individual rgdps eg pAbs expressing the desired specificity eg for anantigen, can be isolated from the complex library using the conventionalscreening techniques (e.g. as described in Harlow, E., and Lane, D.,1988, supra Gherardi, E et al. 1990. J. Immunol. meth. 126 p 61-68).

The applicants have also devised a series of novel selection techniquesthat are practicable only because of the unique properties of rgdps. Thegeneral outline of some screening procedures is illustrated in FIG. 2(i) and FIG. 2( ii) using pAbs as an example type of rgdp.

The population/library of pAbs to be screened could be generated fromimmunised or other animals; or be created in vitro by mutagenisingpre-existing phage antibodies (using techniques well-known in the artsuch as oligonucleotide directed mutagenesis (Sambrook, J., et al., 1989Molecular Cloning a Laboratory Manual, Cold Spring Harbor LaboratoryPress). This population can be screened in one or more of the formatsdescribed below with reference to FIG. 2( i) and FIG. 2( ii), to derivethose individual pAbs whose antigen binding properties are differentfrom sample c.

Binding Elution

FIG. 2( i) shows antigen (ag) bound to a solid surface (s) the solidsurface (s) may be provided by a petri dish, chromatography beads,magnetic beads and the like. The population/library of pAbs is thenpassed over the ag, and those individuals p that bind are retained afterwashing, and optionally detected with detection system d. A detectionsystem based upon anti-fd antisera is illustrated in more detail belowin example 4. If samples of bound population p are removed underincreasingly stringent conditions, the binding affinity represented ineach sample will increase. Conditions of increased stringency can beobtained, for example, by increasing the time of soaking or changing thepH of the soak solution, etc.

Competition

Referring to FIG. 2( ii) antigen ag can be bound to a solid support sand bound to saturation by the original binding molecule c. If apopulation of mutant pAb (or a set of unrelated pAbs) is offered to thecomplex, only those that have higher affinity for antigen ag than c willbind. In most examples, only a minority of population c will bedisplaced by individuals from population p. If c is a traditionalantibody molecule, all bound material can be recovered and bound precovered by infecting suitable bacteria and/or by use of standardtechniques such as PCR.

An advantageous application is where ag is used as a receptor and c thecorresponding ligand. The recovered bound population p is then relatedstructurally to the receptor binding site/and or ligand. This type ofspecificity is known to be very useful in the pharmaceutical industry.

Another advantageous application is where ag is an antibody and c itsantigen. The recovered bound population p is then an anti-idiotypeantibody which have numerous uses in research and the diagnostic andpharmaceutical industries.

At present it is difficult to select directly for anti-idiotypeantibodies. pAbs would give the ability to do this directly by bindingpAb libraries (eg a naive library) to B cells (which express antibodieson their surface) and isolating those phage that bound well.

In some instances it may prove advantageous to pre-select population p.For example, in the anti-idiotype example above, p can be absorbedagainst a related antibody that does not bind the antigen.

However, if c is a pAb, then either or both c and p can advantageouslybe marked in some way to both distinguish and select for bound p overbound c. This marking can be physical, for example, by pre-labelling pwith biotin; or more advantageously, genetic. For example, c can bemarked with an EcoB restriction site, whilst p can be marked with anEcoK restriction site (see Carter, P. et al., 1985, Nucl. Acids Res. 13,4431-4443). When bound p+c are eluted from the antigen and used toinfect suitable bacteria, there is restriction (and thus no growth) ofpopulation c (i.e. EcoB restricting bacteria in this example). Any phagethat grew, would be greatly enriched for those individuals from p withhigher binding affinities. Alternatively, the genetic marking can beachieved by marking p with new sequences, which can be used tospecifically amplify p from the mixture using PCR.

Since the bound pAbs can be amplified using for example PCR or bacterialinfection, it is also possible to rescue the desired specificity evenwhen insufficient individuals are bound to allow detection viaconventional techniques.

The preferred method for selection of a phage displaying a proteinmolecule with a desired specificity or affinity will often be elutionfrom an affinity matrix with a ligand (eg example 21). Elution withincreasing concentrations of ligand should elute phage displayingbinding molecules of increasing affinity. However, when eg a pAb bindsto its antigen with high affinity or avidity (or another protein to itsbinding partner) it may not be possible to elute the pAb from anaffinity matrix with molecule related to the antigen. Alternatively,there may be no suitable specific eluting molecule that can be preparedin sufficiently high concentration. In these cases it is necessary touse an elution method which is not specific to eg the antigen-antibodycomplex. Some of the non-specific elution methods generally used reducephage viability for instance, phage viability is reduced with time atpH12 (Rossomando, E. F. and Zinder N. D. J. Mol. Biol. 36 387-399 1968).There may be interactions between eg antibodies and affinity matriceswhich cannot be disrupted without completely removing phage infectivity.In these cases a method is required to elute phage which does not relyon disruption of eg the antibody-antigen interaction. A method wastherefore devised which allows elution of bound pAbs under mildconditions (reduction of a dithiol group with dithiothreitol) which donot disrupt phage structure (example 47).

This elution procedure is just one example of an elution procedure undermild conditions. A particularly advantageous method would be tointroduce a nucleotide sequence encoding amino acids constituting arecognition site for cleavage by a highly specific protease between theforeign gene inserted, in this instance a gene for an antibody fragment,and the sequence of the remainder of gene III. Examples of such highlyspecific proteases are Factor X and thrombin. After binding of the phageto an affinity matrix and elution to remove non-specific binding phageand weak binding phage, the strongly bound phage would be removed bywashing the column with protease under conditions suitable for digestionat the cleavage site. This would cleave the antibody fragment from thephage particle eluting the phage. These phage would be expected to beinfective, since the only protease site should be the one specificallyintroduced. Strongly binding phage could then be recovered by infectingeg. E. coli TG1 cells.

An alternative procedure to the above is to take the affinity matrixwhich has retained the strongly bound pAb and extract the DNA, forexample by boiling in SDS solution. Extracted DNA can then be used todirectly transform E. coli host cells or alternatively the antibodyencoding sequences can be amplified, for example using PCR with suitableprimers such as those disclosed herein, and then inserted into a vectorfor expression as a soluble antibody for further study or a pAb forfurther rounds of selection.

Another preferred method for selection according to affinity would be bybinding to an affinity matrix containing low amounts of ligand.

If one wishes to select from a population of phages displaying a proteinmolecule with a high affinity for its ligand, a preferred strategy is tobind a population of phage to an affinity matrix which contains a lowamount of ligand. There is competition between phage, displaying highaffinity and low affinity proteins, for binding to the ligand on thematrix. Phage displaying high affinity protein is preferentially boundand low affinity protein is washed away. The high affinity protein isthen recovered by elution with the ligand or by other procedures whichelute the phage from the affinity matrix (example 35 demonstrates thisprocedure).

In summary then, for recovery of the packaged DNA from the affinitystep, the package can be simply eluted, it can be eluted in the presenceof a homologous sbp member which competes with said package for bindingto a complementary sbp member; it could be removed by boiling, it couldbe removed by proteolytic cleavage of the protein; and other methodswill be apparent to those skilled in the art eg. destroying the linkbetween the substrate and complementary sbp member to release saidpackaged DNA and sbp member. At any rate, the objective is to obtain theDNA from the package so that it can be used directly or indirectly, toexpress the sbp member encoded thereby.

The efficiency of this selection procedure for pAbs and the ability tocreate very large libraries means that the immunisation techniquesdeveloped to increase the proportion of screened cells producingantibodies of interest will not be an absolute requirement. Thetechnique allows the rapid isolation of binding specificities egantigen-binding specificities, including those that would be difficultor even unobtainable by conventional techniques, for example, catalyticor anti-idiotypic antibodies. Removal of the animal altogether is nowpossible, once a complete library of the immune repertoire has beenconstructed.

The novel structure of the pAb molecule can be used in a number of otherapplications, some examples of which are:

Signal Amplification

Acting as a novel molecular entity in itself, rgdps eg pAbs combine theability to bind a specific molecule eg antigen with amplification, ifthe major coat protein is used to attach another moiety. This moiety canbe attached via immunological, chemical, or any other means and can beused, for example, to label the complex with detection reagents orcytotoxic molecules for use in vivo or in vitro.

Physical Detection

The size of the rgdps eg pAbs can be used as a marker particularly withrespect to physical methods of detection such as electron microscopyand/or some biosensors, e.g. surface plasmon resonance.

Diagnostic Assays

The rgdps eg pAbs also have advantageous uses in diagnostic assays,particularly where separation can be effected using their physicalproperties for example centrifugation, filtration etc.

In order that the invention is more fully understood, embodiments willnow be described in more detail by way of example only and not by way oflimitation with reference to the figures described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the basic structure of the simplest antibody molecule IgG.

FIGS. 2( i) and 2(ii) show schematically selection techniques whichutilise the unique properties of pAbs; 2(i) shows a binding/elutionsystem; and (2 ii) shows a competition system (p=pAb; ag=antigen towhich binding by pAb is required; c=competitor population e.g. antibody,pAb, ligand; s=substrate (e.g. plastic beads etc); d=detection system.

FIG. 3 shows the vector fd-tet and a scheme for the construction ofvectors, fdTPs/Bs (for insertion of VH coding sequences) and fdTPs/Xhfor the insertion of scFv coding sequences.

FIGS. 4( i)-4(ii) show the nucleotide sequences for the oligonucleotidesand vectors. All sequences are drawn 5′ to 3′ and are numbered accordingto Beck et al., 1978, Nucl. Acid Res., 5: 4495-4503. 4(i) shows thesequences of the oligonucleotides used for mutagenesis (oligo's 1 and 2)or sequencing (oligo 3). The sequences shown were synthesized on anApplied Biosystems, oligonucleotide synthesizer and are complementary tothe single stranded form of fd-tet (they are in the anti-sense form withrespect to gene III). 4(ii) shows the sequences of the variousconstructs around the gene III insertion site. These sequences are drawnin the sense orientation with respect to gene III; (A) fd-tet (andfdTδBst) (B) fdTPs/Bs and (C) fdTPs/Xh. The key restriction enzyme sitesare shown along with the immunoglobulin amino acids contributed by thevectors, (amino acid single letter code is used, see Harlow, E., andLane, D., 1988 supra.).

FIG. 5 shows the nucleotide and amino acid sequences for scFv in thevector scFvD1.3 myc. This gives the sequence of the anti-lysozyme singlechain Fv and surrounding sequences in scFvD1.3 myc, showing theN-terminal pel B signal peptide sequence and the C-terminal myc tagsequence (Ward, E. S., et al., 1989, supra.). Also shown is the peptidesequence linking the VH and VL regions. The amino acid sequence isrepresented above the nucleotide sequence by the single letter code, seeHarlow, E., and Lane D., 1988 supra.

FIG. 6 shows the binding of pAbs to lysozyme and the effect of varyingthe amount of supernatant. Each point is the average of duplicatesamples. Lysozyme was coated at 1 mg/ml in 50 mM NaHCO₃.

FIG. 7 shows the effect of varying the coating concentration of lysozymeor bovine serum albumin on the binding of pAbs to lysozyme in graphicalform. Each point is the average of duplicate samples.

FIG. 8 shows the sequence around the cloning site in gene III offd-CAT2. Restriction enzyme sites are shown as well as the amino acidsencoded by antibody derived sequences. These are flanked at the 5′ endby the gene III signal peptide and at the 3′ end by 3 alanine residues(encoded by the Not 1 restriction site) and the remainder of the maturegene III protein. The arrow shows the cleavage site for cutting of thesignal peptide.

FIG. 9 shows the binding of pAb (1.3) to lysozymes. Binding of phage asdetected by ELISA to (a) hen egg-white lysozyme (HEL) (b) turkeyegg-white lysozyme (TEL), (c) human lysozyme (HUL), (d) bovine serumalbumin (BSA). A further control of (e) fdTPs/Bs to HEL.

FIG. 10 shows a map of FabD1.3 in pUC19.

FIG. 11 shows the ELISA results providing a comparison oflysozyme-binding by phage-Fab and phage-scFv. Vector=fdCAT2 (example 5);fdscFv(OX)=pAbNQ11 (Example 9); fdVHCH1 (D1.3)=grown in normal cells(i.e. no L chain, see example 7); fdFab(D1.3) i.e. fdVHCH1 (D1.3) grownin cells containing D1.3 L chain; fdscFv (D1.3)=pAbD1.3.

FIGS. 12 a-12 b show oligonucleotide probing of affinity purified phage.10¹² phage in the ratio of 1 pAb (D1.3) in 4×10⁴ fdTPS/Bs phages wereaffinity purified and probed with an oligonucleotide specific for pAb(D1.3) A is a filter after one round of affinity purification (900colonies total) and B is a filter after two rounds (372 colonies total).

FIG. 13 shows the sequence of the anti-oxazolone antibody fragment NQ11scFv. The sequence contributed by the linker is shown in the lower case.The sequence for VH is before the linker sequence and the sequence forVL is after the linker.

FIG. 14 shows the ELISA results for binding pAb NQ11 and pAb D1.3 andvector fdTPs/xh to specified antigens.

FIG. 15 shows the sequence surrounding the phoA insertion infd-phoAla166. The restriction sites used for cloning are shown, as wellas the amino acids encoded by phoA around the insertion site. The firstfive amino acids of the mature fusion come from gene III.

FIG. 16( i) shows the structure of gene III and the native BamHI siteinto which a scFv coding sequence was inserted in example 13 and FIG.16( ii) shows the natural peptide linker sites A and B for possibleinsertion of scFv coding sequences.

FIG. 17 shows schematically the protocol for PCR assembly of mouse VHand VLK repertoires for phage display described in example 14.

FIG. 18 shows examples of the final products obtained with the procedureof example 14. Lanes a and b show the products of the initial PCR usingheavy and light chain primers respectively; lane c shows the completeassembled 700 bp product before final digestion with Not1 and ApaL1; M1,M2 markers Φ174 Hae III digest and 123 base pair ladder (BRL Limited,P.O. Box 35, Washington Road, Paisley, Scotland) respectively.

FIG. 19 shows the binding of ¹²⁵I-PDGF-BB to fd h-PDGFB-R phage inimmunoprecipitation assay and comparison to fdTPs/Bs and no phagecontrols; binding is expressed as a percentage of the total ¹²⁵I-PDGF-BBadded to the incubation.

FIG. 20 shows the displacement of ¹²⁵I-PDGF-BB bound to fd-h-PDGFB-Rphage using unlabelled PDGF-BB measured using an immunoprecipitationassay. Binding is expressed as a percentage of the total ¹²⁵I-PDGF-BBadded to the incubation.

FIG. 21 shows the displacement of ¹²⁵I-PDGF-BB bound to fd-h-PDGFB-Rphage using unlabelled PDGF-BB measured using an immunoprecipitationassay. Non-specific binding of ¹²⁵I-PDGF-BB to vector phage fdTPs/Bs inthe absence of added unlabelled PDGF was deducted from each point.

FIG. 22 shows the results of an ELISA of lysozyme binding by pCAT-3 scFvD1.3 phagemid in comparison with pCAT-3 vector (both rescued by M13K07)and fdCAT2 scFv D1.3 as described in example 17. The ELISA was performedas described in example 6 with modifications detailed in example 18.

FIG. 23( i)-FIG. 23( ii) show the digestion pattern seen when individualclones, selected at random from a library of single chain Fv antibodygenes derived from an immunised mouse; are digested with BstN1.

FIG. 24 shows VH and VK gene sequences derived from the combinatoriallibrary in example 21 and the hierarchical library in example 22.

FIG. 25 shows a matrix of ELISA signals for clones derived from randomcombinatorial library. Designation of the clones is as in FIG. 24. Thenumber of clones found with each combination is shown by the numerals.

FIG. 26A shows the phagemid pHEN1a derivative of pUC119 described inexample 24; and the cloning sites in the phagemid pHEN.

FIG. 27. The antibody constructs cloned into fd-CAT2 and pHEN1 fordisplay on the surface of phage. Constructs I, II, III and IV werecloned into both fd-CAT2 (as ApaLI-NotI fragments) and pHEN1 (asSfiI-NotI fragments) and pHEN1 (as SfiI-NotI fragments). All theconstructs contained the heavy chain (VH) and light chain (VK) variableregions of the mouse anti-phOx antibody NQ10.12.5. The constant domainswere human CK and CH1 (γ1 isotype).

FIG. 28. Three ways of displaying antibody fragments on the surface ofphage by fusion to gene III protein.

FIG. 29. Western blot of supernatant taken from pHEN1-II(+) or pHEN1(−)cultures in E. coli HB2151, showing secretion of Fab fragment frompHEN1-II only. The anti-human Fab detects both H and L chain. Due to theattached c-myc tag, the L chain, highlighted by both anti-c-myc tag andanti-human CK antisera, is slightly larger (calculated Mr 24625) thanthe H chain (calculated Mr23145).

FIG. 30 is a plot showing the effect of lysozyme dilution on ratio ofELISA signals obtained using pAbD1.3 or soluble scFv D1.3.

FIG. 31 is a plot showing the effect of lysozyme dilution on ELISAsignals obtained using fdTscFvD1.3 and soluble scFvD1.3.

FIG. 32 is a plot showing positive results from an ELISA screen of phagedisplaying scFv fragments derived from the cell line 013 which express amonoclonal antibody directed against oestriol.

FIG. 33 is a plot showing positive results from an ELISA screen of phagedisplaying scFv fragments derived from the cell line 014 which express amonoclonal antibody directed against oestriol.

FIG. 34 is a Western Blot showing expression of the alkalinephosphatase-gene 3 fusion. 16 μl of 50 fold concentrate of each phagesample was detected on western blots with either anti-gene 3 antiserum(e-f) or with anti-alkaline phosphatase antiserum (c-f)

a) fd-phoAla166 grown in TG1 cells

b) fd-phoAla166 grown in KS272 cells

c) fdCCAT2 grown in TG1 cells

d) fdCAT2 grown in TG1 cells, mixed with 13 ng of purified alkalinephosphatase

e) fd-phoAla166 grown in TG1 cells

f) fdCAT2 grown in TG1 cells.

FIGS. 35A-35B are Western Blots showing ultrafiltration of phage-enzyme100 μl of 50 fold concentrate of phage (representing 5 mls of culturesupernatant) was centrifuged through ultrafiltration membranes withnominal molecular weight retention of 300,000 daltons. Western blots offlow through and retentate fractions were detected with anti-alkalinephosphatase antiserum. The equivalent of 800 μl of original culturesupernatant was run on the gel.

FIG. 35A. Phage were grown in TG1 cells. a) fd-phoAla166 beforeultrafiltration (short exposure). b) fd-phoAla166 beforeultrafiltration. c) fd-phoAla166 material retained on ultrafiltrationmembrane.

FIG. 35B. Phage were grown in KS272 cells. a) fd-phoAla166 beforeultrafiltration. b) fd-phoAla166 material retained on ultrafiltrationmembrane. c) fdCAT2. d) fdCAT2 mixed with purified alkaline phosphatasebefore ultrafiltration. e) Retentate from sample d. f) Flow through fromsample d.

FIG. 36 Electrophoresis of samples from stages of a Fab assembly.Samples from different stages in the PCR Fab assembly process describedin example 33 were subjected to electrophoresis on a 1% TAE-agarose gel.Samples from a comparable scFv assembly process (as in example 14) areshown for comparison. Samples left to right are:

M = Markers VHCH1 = sequences encoding VHCH1 domains amplified by PCRVKCK = sequences encoding VKCK domains amplified by PCR −L = Fabassembly reaction performed in absence of linker +L = Fab PCR assemblyreaction product VHCH1 plus VKCK plus linker M = Markers VK = sequencesencoding VK domain amplified by PCR VL = sequences encoding VH domainsamplified by PCR −L = scFv assembly reaction in absence of linker +L =scFv assembly reaction in presence of linker M = Markers

FIG. 37. Comparison of ELISA signals with scFv D1.3 cloned in fd-CAT2(fd) or pCAT-3. pCAT-3 scFv1.3 has been rescued with M13K07 (KO7).M13KO7ΔgIII No 3 (gIII No 3) or M13KO7 gIIIΔNo 2 (g111No2). Phageantibodies are compared at 10 times (10×) 1 times (1×) or 0.1 times(0.1×) concentrations relative to concentration in the supernatant afterovernight growth. The fdCAT2 and pCAT-3 non-recombinant vector signalswere <0.01 at 10× concentration. M13KO7 gIIIΔNo 1 did not rescue at all,as judged by no signal above background in this ELISA.

FIGS. 38A-38B. Western blot of PEG precipitated phage used in ELISAprobed with anti-g3p. Free g3p and the g3p-scFvD1.3 fusion bands arearrowed.

Sample 1—fd scFvD1.3

Sample 2—pCAT3 vector

Sample 3—pCAT3 scFvD1.3 rescued with M13KO7, no IPTG

Sample 4—pCAT3 scFvD1.3 rescued with M13KO7, 50 μM IPTG

Sample 5—pCAT3 scFvD1.3 rescued with M13KO7, 100 μM

-   -   IPTG        Sample 6—pCAT3 scFvD1.3 rescued with M13KO7 gIIIΔ No3    -   (no IPTG)        Sample 7—pCAT3 scFvD1.3 rescued with M13KO7 gIIIΔ No 2    -   (no IPTG)

FIG. 38A samples contain the equivalent of 8 μl of phagemid culturesupernatant per track, and 80 μl of the fd supernatant (10-fold lowerphage yield than the phagemid). FIG. 38B phagemid samples are those usedin FIG. 38A at a five-fold higher sample loading (equivalent to 40 μl ofculture supernatant per track) to enable visualisation of the fusionband in samples rescued with parental M13K07.

FIG. 39 is a graph showing fdCAT2scFvD1.3 enrichment produced from amixture of fdCAT2scFvD1.3 and fdCAT2TPB4 by one round of panning.

FIG. 40 is a graph showing fdCAT2scFvD1.3 enrichment produced from amixture of fdCAT2scFvD1.3 and fdCAT2TPB1 by one round of panning.

FIG. 41. Western blot of phage proteins of fdCAT2(1) and fd-tet-SNase(2)with anti-g3p antiserum. Marker molecular weights bands are indicated(kD).

FIG. 42. Nuclease assay of soluble SNase (3 ng)(A-1), fd-tet-SNase(4×10⁹TU, (B-1), fd-CAT2(2×10¹⁰TU)(C-1) and of a PEG-precipitated fdCAT2and SNase mixture (2×10¹⁰TU and 0.7 ug) (D-1) in a 10-fold dilutionseries (1 to 3 or 4). Marker (M) is a HindIII digest of λ-DNA (NewEngland Biolabs).

FIG. 43. ELISA signals obtained with fd-tet, fd-CD4-V1 and fd-CD4-V1V2.In each group of three, the samples are left to right phage concentrate(SN); phage concentrate plus soluble CD4(SN+sCD4); phage concentrateplus gp 120 (SN+gp 120).

FIGS. 44( i)-(ii) show the DNA sequence of scFv B18 (anti-NP).

FIG. 45 shows a map of the insert of sequences encoding FvD1.3 presentin fd-tet FvD1.3 (example 39). rbs designates the ribosome binding site.Gene III is now shown in its full length.

FIG. 46. shows an ELISA assay of phages displaying FvD1.3 or scFvD1.3 bybinding to plates coated with lysogyme. Signals obtained at variousdilution factors are shown. FvD1.3 (ΔS-Stuffer) which does not expressFv was used as a control.

FIG. 47. shows a schematic representation of steps involved in the PCRassembly of nucleotide sequences encoding human Fab fragments. Detailsare in example 40.

FIG. 48( i) shows a map of plasmid pJM1-FabD1.3 which is used for theexpression of soluble human Fab fragments and as a template for thesynthesis of linker DNA for Fab assembly. FIG. 48( ii) is a schematicrepresentation of sequences encoding a Fab construct. FIG. 48( iii)shows the sequence of DNA template for the synthesis of linker DNA forFab assembly.

FIG. 49. shows a schematic representation of steps involved in the PCRassembly of nucleotide sequences encoding human scFv fragments. Detailsare in example 42.

FIGS. 50( i)-(ii). ELISA assay of phage antibodies using plates coatedwith turkey egg lysogyme. Two clones B1 and A4 are shown derived bymutagenesis and selection from pAbD1.3 (example 45). Concentration (xaxis) refers to the concentration of phage for each sample relative tothe concentration in culture supernatant. B1 has raised binding toturkey egg lysogyme compared to D1.3. A4 has reduced binding to hen egglysogyme compared to D1.3.

FIG. 51. ELISA of phage antibodies binding to HEL and TEL. Clone 1 isfdCAT2scFvD1.3. Clones 2 to 10 were obtained from the library (example46) after selection. The background values as defined by binding ofthese clones to BSA were subtracted.

FIG. 52. shows the DNA sequence of the light chains D1.3 M1F and M21derived by selection from a hierarchical library in example 46.

FIG. 53 shows a Fv lambda expression vector (example 48) derived frompUC119. It contains the rearranged lambdal germ line gene. The heavy andlight chain cassettes each contain a ribosome binding site upstream ofthe pel B leader (Restriction sites shown as: H=Hind III; Sp=SphI;B=BamHI, E=EcoRI.

MATERIALS AND METHODS

The following procedures used by the present applicants are described inSambrook, J. et al., 1989 supra.: restriction digestion, ligation,preparation of competent cells (Hanahan method), transformation,analysis of restriction enzyme digestion products on agarose gels,purification of DNA using phenol/chloroform, 5′-end labelling ofoligonucleotides, filter screening of bacterial colonies, preparation of2×TY medium and plates, preparation of tetracycline stock solutions,PAGE of proteins, preparation of phosphate buffered saline.

All enzymes were supplied by New England Biolabs (CP Laboratories, POBox 22, Bishop's Stortford, Herts., England) and were used according tomanufacturer's instructions unless otherwise stated.

The vector fd-tet (Zacher, A. N. et al., 1980, supra) was obtained fromthe American Type Culture Collection (ATCC No. 37000) and transformedinto competent TG1 cells (genotype: K₁₂₅ (lac-pro), sup E, thi, hsdD5/FtraD36, pro A+B+, Lac 1^(q), lac δM15).

Viral particles were prepared by growing TG1 cells containing thedesired construct in 10 to 100 mls 2×TY medium with 15 μg/mltetracycline for 16-24 hours. The culture supernatant was collected bycentrifugation for 10 mins at 10,000 rpm in an 8×50 ml rotor, SorvalRC-5B centrifuge. Phage particles were precipitated by adding ⅕th volume20% polyethylene glycol (PEG)/2.5M NaCl and leaving at 4° C. for 1 hour.These were spun for 15 minutes as described above and the pelletsresuspended in 10 mM Tris/HCl pH 8, 1 mM EDTA to 1/100th of the originalvolume. Residual bacteria and undissolved material were removed byspinning for 2 minutes in a microcentrifuge. Single stranded DNA formutagenesis or sequencing was prepared from concentrated phage accordingto Sambrook, J., et al., 1989, supra.

INDEX OF EXAMPLES Example 1 Design of Insertion Point Linkers andConstruction of Vectors

This example covers the construction of two derivatives of the phagevector fd-tet: a) fdTPs/Bs for the insertion of VH coding sequences; andb) fdTPs/Xh for the insertion of scFv coding sequences. The derivativevectors have a new BstEII site for insertion of sequences.

Example 2 Insertion of Immunoglobulin Fv Domain into Phage

This example covers the insertion of scFv coding sequences derived froman anti-lysozyme antibody D1.3 into fdTPs/Xh to give the constructfdTscFvD1.3.

Example 3 Insertion of Immunoglobulin VH Domain into Phage

This example covers the insertion of VH coding sequences derived from ananti-lysozyme antibody D1.3 into fdTPs/Bs to give the constructfdTVHD1.3.

Example 4 Analysis of Binding Specificity of Phage Antibodies

This example investigates the binding specificities of the constructsfdTscFvD1.3 and fdTVHD1.3.

Example 5 Construction of fdCAT2

This example covers the construction of the derivative fdCAT2 of thephage vector fdTPs/Xh. The derivative has restriction sites for enzymesthat cut DNA infrequently.

Example 6 Specific Binding of Phage Antibody (pAb) to Antigen

This example shows the binding of pAb fdTscFvD1.3 to lysozyme by ELISA.

Example 7 Expression of FabD1.3

This example concerns the display of an antibody Fab fragment at thephage surface. The VH-CH₁ chain is expressed by fdCAT2. The VL-CL chainis expressed by pUC19 in a bacterial host cell also infected withfdCAT2.

Example 8 Isolation of Specific, Desired Phage from a Mixture of VectorPhage

This example shows how a phage (e.g. fdTscFvD1.3) displaying a bindingmolecule can be isolated from vector phage by affinity techniques.

Example 9 Construction of pAb Expressing Anti-Hapten Activity

This example concerns the insertion of scFv coding sequences derivedfrom the anti-oxazolone antibody NQ11 into fdTPs/Xh to generate theconstruct pAbNQ11. The example shows the binding of pAbNQ11 to oxazaloneby ELISA.

Example 10 Enrichment of pAbD1.3 from Mixtures of Other pAbs by AffinityPurification

This example shows how a phage (eg. pAbD1.3) displaying one sort ofbiding molecule can be isolated from phage (e.g. pAbNQ11) displayinganother sort of binding molecule by affinity techniques.

Example 11 Insertion of a Gene Encoding an Enzyme (Alkaline Phosphate)into fdCAT2

This example concerns the invention of coding sequences for an enzymeinto the vector fdCAT2 to give the phage enzyme, fdphoAla116.

Example 12 Measuring Enzyme Activity Phage—Enzyme

This example shows the functionality of an enzyme (alkaline phosphatase)when displayed at the phage surface (fdphoAla166).

Example 13 Insertion of Binding Molecules into Alternative Sites in thePhage

This example covers the insertion of scFv coding sequences derived froma) the anti-lysozyme antibody D1.3; and b) the anti-oxazalone antibodyNQ11 into a BamHI site of fdTPs/Xh to give the constructs fdTBam1 havingan NQ11 insert.

Example 14 PCR Assembly of Mouse VH and VLK Repertoires for PhageDisplay

This example concerns a system for the display on phage of all VH andVLK repertoires encoded by a mouse. The system involves the followingsteps. 1) Preparation of RNA from spleen. 2) Preparation of cDNA fromthe RNA 3) Use of primers specific for antibody sequences to PCR amplifyall VH and VLK cDNA coding sequences 4) Use of PCR to create a linkermolecule from linking pairs of VH and VLK sequences 5) Use of PCR toassemble continuous DNA molecules each comprising a VH sequence, alinker and a VLK sequence. The specific VH/VLK combination is randomlyderived 6) Use of PCR to introduce restriction sites.

Example 15 Insertion of the Extracellular Domain of a Human Receptor forPlatelet Derived Growth Factor (PDGF) Isoform BB into fdCAT2

This example concerns the insertion of coding sequences for theextracellular domain of the human receptor for PDGF into the vectorfdCAT2 to give the construct fdhPDGFBR.

Example 16 Binding of 125 I-PDGF-BB to the Extracellular Domain of theHuman Receptor for PDGF Isoform BB Displayed on the Surface of fd Phage.Measured Using an Immunoprecipitation Assay

This example shows that the human receptor PDGF Isoform BB is displayedon the surface of the phage in a form which has the ability to bind itsligand.

Example 17 Construction of Phagemid Containing Gene III Fused with theCoding Sequence for a Binding Molecule

This example concerns the construction of two phagemids based on pUC119which separately contain gene III from fdCAT2 and the gene III scFvfusion fdCAT2seFvDI.3 to generate pCAT2 and pCAT3 scFvDI.3 respectively.

Example 18 Rescue of Anti-Lysozyme Antibody Specificity frompCAT3scFvD1.3 by M13KO7

This example describes the rescue of the coding sequence for the geneIIIscFv fusion from pCAT3scFvD1.3 by M13M07 helper phage growth, phagewere shown to be displaying scFv anti-lypozyme activity by ELISA.

Example 19 Transformation Efficiency of PCAT-3 and pCAT-3 scFvD1.3Phagemids

This example compared the efficiency of the phagemids pVC119, pCAT-3 andpCAT3scFvD1.3 and the phage fdCAT2 to transform E. coli.

Example 20 PCR Assembly of a Single Chain Fv Library from an ImmunisedMouse

This example concerns a system for the display on phage of scFv(comprising VH and VL) from an immunised mouse using the basic techniqueoutlined in example 14 (cDNA preparation and PCR assembly of the mouseVH and VLK repertoires) and ligating the PCR assembled sequences intofdCAT2 to create a phage library of 10⁵ clones. Testing of 500 clonesshowed that none showed specificity against phox.

Example 21 Selection of Antibodies Specific for 2-Phenyl-5-Oxazolonefrom a Repertoire from an Immunised Mouse

This example shows that phage grown from the library established inexample 20 can be subjected to affinity selection using phOX to selectthose phage displaying scFv with the desired specificity.

Example 22 Generation of Further Antibody Specificities by the Assemblyof Hierarchical Libraries

This example concerns the construction of hierarchical libraries inwhich a given VH sequence is combined with the complete VLK repertoireand a given VLK sequence is combined with the complete VH repertoire andselection from these libraries of novel VH and VL pairings.

Example 23 Selection of Antibodies Displayed on Bacteriophage withDifferent Affinities for 2-phenyl-5-Oxazolone Using AffinityChromatography

This example concerns the separation by affinity techniques of phagesdisplaying scFv fragments with differing binding affinities for a givenantigen.

Example 24 Construction of Phagemid pHEN1 for the Expression of AntibodyFragments Expressed on the Surface of Bacteriophage FollowingSuperinfection

This example concerns the construction of the phagemid pHEN1 derivedfrom pUC119. pHEN1 has the features shown in FIG. 26.

Example 25 Display of Single Chain Fv and Fab Fragments Derived from theAnti-Oxazolone Antibody NO 10.12.5 on Bacteriophage fd Using pHEN1 andfdCAT2

This example describes the display of scFv and Fab fragment with aspecificity against phOx on the surface of a bacteriophage. For displayof scFv the phagemid pHEN1 comprises the sequences encoding scFv (VH andVL) for rescue by either the phages VSM13 or fdCAT2. For display of Fabthe phage fdCAT2 comprises the sequence for either the H or L chain as afusion with g3p and the phagemid pHEN1 comprises the sequence for theappropriate H or L chain partner.

Example 26 Rescue of Phagemid Encoding a Gene III Protein Fusion withAntibody Heavy or Light Chains by Phage Encoding the ComplementaryAntibody Displayed on Phage and the Use of this Technique to Make DualCombinatorial Libraries

This example covers the use of phage antibodies encoding the antibodyheavy or light chain to rescue a phagemid encoding a gene 3 proteinfusion with the complementary chain and the assay of Fab fragmentsdisplayed on phage in ELISA. The use of this technique in thepreparation of a dual combinatorial library is discussed.

Example 27 Induction of Soluble scFv and Fab Fragments Using PhagemidpHEN1

This example covers the generation of soluble scFv and Fab fragmentsfrom gene III fusions with sequences encoding these fragments byexpression of clones in pHEN1 in an E. coli strain which does notsuppress amber mutations.

Example 28 Increased Sensitivity in ELISA of Lysozyme Using fdTscFvD1.3as Primary Antibody Compared to Soluble scFvD1.3

This example covers the use of fdTscFvD1.3 in ELISA showing that loweramounts of lysozyme can be detected with phage antibody fdTscFvD1.3 thanwith soluble scFvD1.3.

Example 29 Direct Rescue and Expression of Mouse Monoclonal Antibodiesas Single Chain Fv Fragments on the Surface of Bacteriophage fd

This example covers the display on phage as functional scFv fragments oftwo clones directly derived from cells expressing monoclonal antibodiesdirected against oestriol. Both clones were established to be functionalusing ELISA.

Example 30 Kinetic Properties of Alkaline Phosphatase Displayed on theSurface of Bacteriophage fd

This example concerns the demonstration that the kinetic properties ofan enzyme, alkaline phosphatase, displayed on phage are qualitativelysimilar to those of the same enzyme when in solution.

Example 31 Demonstration Using Ultrafiltration that Cloned AlkalinePhosphatase Behaves as Part of the Virus Particle

This example concerns the construction of the phage enzyme fdphoArg166and the demonstration that both the fusion protein made and thecatalytic activity observed derive from the phage particle.

Example 32 Affinity Chromatography of Phage Alkaline Phosphatase

This example concerns the binding of alkaline phosphatase displayed onphage to an arsenate-Sepharose affinity column and specific elution ofthese phage using the reaction product, phosphate.

Example 33 PCR Assembly of DNA Encoding the Fab Fragment of an AntibodyDirected Against Oxazolone

This example covers the construction of a DNA insert encoding a Fabfragment by separate amplification of heavy and light chain DNAsequences followed by assembly. The construct was then inserted into thephage vector fdCAT2 and the phagemid vector pHEN1 and the Fab fragmentdisplayed on the surface was shown to be functional.

Example 34 Construction of a Gene III Deficient Helper Phage

This example describes the construction of a helper phage derived fromM13KO7 by deleting sequences in gene III. Rescue of pCAT3-scFvD1.3 isdescribed. The scFvD1.3 is expressed at a high level as a fusion usingthe deletion phage, equivalent to expression using fdCAT2-scFvD1.3.

Example 35 Selection of Bacteriophage Expressing scFv Fragments DirectedAgainst Lysozyme from Mixtures According to Affinity Using a PanningProcedure

This example concerns the selection of bacteriophage according to theaffinity of the scFv fragment directed against lysozyme which isexpressed on their surface. The phage of different affinities were boundto Petri dishes coated with lysozyme and, following washing, bound phageeluted using triethylamine. Conditions were found where substantialenrichment could be obtained for a phage with a 5-fold higher affinitythan the phage with which it was mixed.

Example 36 Expression of Catalytically Active Staphylococcal Nuclease onthe Surface of Bacteriophage fd

This example concerns the construction of a phage enzyme which expressesStaphylococcal nuclease and the demonstration that the phage enzymeretains nuclease activity.

Example 37 Display of the Two Aminoterminal Domains of Human CD4 on theSurface of fd Phage

This example covers the cloning of genes for domains of CD4, a cellsurface receptor and member of the immunoglobulin superfamily, intobacteriophage fd. The receptor is shown to be functional on the surfaceof phage by binding to the HIV protein gp120.

Example 38 Generation and Selection of Mutants of anAnti-4-Hydroxy-3-Nitrophenylacetic Acid (NP) Antibody Expressed on PhageUsing Mutator Strains

This example covers the introduction of mutations into a gene for anantibody cloned in phage by growth of the phage in strains whichrandomly mutate DNA due to defects in DNA replication. Several mutationsare introduced into phage which can then be selected from parent phage.

Example 39 Expression of a Fv Fragment on the Surface of Bacteriophageby Non-Covalent Association of VH and VL Domains

This example shows that functional Fv fragments can be expressed on thesurface of bacteriophage by non-covalent association of VH and VLdomains. The VH domain is expressed as a gene III fusion and the VLdomain as a soluble polypeptide. Sequences allowing expression of thesedomains from the anti-lysozyme antibody D1.3 in this form wereintroduced into phage and the resulting displayed Fv fragment shown tobe functional by ELISA.

Example 40 A PCR Based Technique for One Step Cloning of Human V-Genesas Fab Constructs

This example gives methods for the assembly of Fab fragments from genesfor antibodies. Examples are given for genes for antibodies directedagainst Rhesus-D in a human hybridoma and a polyclonal lymphoblasticcell line.

Example 41 Selection of Phage Displaying a Human Fab Fragment DirectedAgainst the Rhesus-D Antigen by Binding to Cells Displaying the Rhesus DAntigen on their Surface

This example concerns the construction of, and display of phageantibodies from, a phagemid encoding a human Fab fragment directedagainst the Rhesus D antigen. Phage displaying this antigen were thenaffinity select d from a background of phage displaying scFvD1.3anti-lysozyme on the basis of binding to Rhesus-D positive red bloodcells.

Example 42 A PCR Based Technique for One Step Cloning of Human scFvConstructs

This example describes the generation of libraries of scFv fragmentsderived from an unimmunized human. Examples are given of the preparationfor phage display of libraries in phagemids of scFv fragments derivedfrom IgG and IgM sequences.

Example 43 Isolation of Binding Activities from a Library of scFvs froman Unimmunized Human

This example describes the isolation, from the library of scFv fragmentsderived from IgM genes of an unimmunized human, of clones for phageantibodies directed against BSA, lysozyme and oxazolone. Selection wasby panning or affinity chromatography and analysis of bindingspecificity by ELISA. Sequencing of the clones showed them to be ofhuman origin.

Example 44 Rescue of Human IgM Library Using Helper Phage Lacking Gene 3(g3)

This example covers the isolation, from the library of scFv fragments ofunimmunized human IgM genes, of clones of phage antibodies of clones forphage antibodies specific for thyroglobulin and oxazolone. In thisexample rescue was with M13K07gIII No3 (NCTC12478), a helper phagedefective in gene III. Fewer rounds of selection appeared necessary fora phagemid library rescued with this phage compared to one rescued withM13K07.

Example 45 Alteration of Fine Specificity of scFvD1.3 Displayed on Phageby Mutagenesis and Selection on Immobilized Turkey Lysozyme

This example covers the in vitro mutagenesis of pCATscFvD1.3 byreplacement, with random amino acids, of residues known to be ofimportance in the preferential recognition of hen egg lysozyme overturkey egg lysozyme by scFvD1.3. Following selection for phageantibodies recognising turkey egg lysozyme by affinity chromatography,clones were analysed for specificity by ELISA. Two groups of clones werefound with more equal recognition of hen and turkey lysozymes, one withincreased ELISA signal with the turkey enzyme and one with reducedsignal for the hen enzyme.

Example 46 Modification of the Specificity of an Antibody by Replacementof the VLK Domain by a VLK Library Derived from an Unimmunised Mouse

This example shows that replacement of the VL domain of scFvD1.3specific for hen eggwhite lysozyme (HEL) with a library of VL domainsallows selection of scFv fragments which bind also to turkey eggwhitelysozyme (TEL). The scFv fragments were displayed on phage and selectionby panning on tubes coated with TEL. Analysis by ELISA showed cloneswith enhanced binding to TEL compared to HEL. Those with highest bindingto TEL were sequenced.

Example 47 Selection of a Phage Antibody Specificity by Binding to anAntigen Attached to Magnetic Beads. Use of a Cleavable Reagent to AllowElution of Bound Phage Under Mild Conditions

This examples covers the use of a cleavable bond in the affinityselection method to allow release of bound phage under mild conditions.pAbNQ11 was enriched approximately 600 fold from a mixture with pAbD1.3by selection using biotinylated Ox-BSA bound to magnetic beads. Thecleavage of a bond between BSA and the biotin allows elution of thephage.

Example 48 Use of Cell Selection to provide an Enriched Pool of AntigenSpecific Antibody Genes, Application to Reducing the Complexity ofRepertoires of Antibody Fragments Displayed on the Surface ofBacteriophage

This example covers the use of cell selection to produce an enrichedpool of genes encoding antibodies directed against4-hydroxy-3-nitrophenylacetic acid and describes how this techniquecould be used to reduce the complexity of antibody repertoires displayedon the surface of bacteriophage.

Example 1 Design of Insertion Point Linkers and Construction of Vectors

The vector fd-tet has two BstEII restriction sites flanking thetetracycline resistance gene (FIG. 3). Since the strategy for insertingthe VH fragments was to ligate them into a newly inserted BstEII sitewithin gene III, it was advantageous to delete the original BstEII sitesfrom fd-tet. This was achieved by digesting fd-tet with the restrictionenzyme BstEII, filling-in the 5′ overhangs and re-ligating to generatethe vector fdTδBst. Digestion of fd-tet with BstEII (0.5 units/μl) wascarried out in 1×KGB buffer (100 mM potassium glutamate, 23 mMTris-acetate (pH 7.5), 10 mM magnesium acetate, 50 μg/ml bovine serumalbumin, 0.5 mM dithiothreitol (Sambrook, J., et al., 1989, supra.) withDNA at a concentration of 25 ng/μl. The 5′ overhang was filled in, using2×KGB buffer, 250 μM each dNTP's (Pharmacia Ltd., Pharmacia House,Midsummer Boulevard, Milton Keynes, Bucks., UK.) and Klenow Fragment(Amersham International, Lincoln Place, Green End, Aylesbury, Bucks.,UK) at 0.04 units/μl. After incubating for 1 hour at room temperature,DNA was extracted with phenol/chloroform and precipitated with ethanol.

Ligations were carried out at a DNA concentration of 50 ng/μl).Ligations were transformed into competent TG1 cells and plated onto TYplates supplemented with 15 μg/ml tetracycline. This selects for vectorswhere the gene for tetracycline resistance protein has reinserted intothe vector during the ligation step. Colonies were picked into 25 mls of2×TY medium supplemented with 15 μg/ml tetracycline and grown overnightat 37° C.

Double stranded DNA was purified form the resulting clones using thegene-clean II kit (Bio101 Inc., PO Box 2284, La Jolla, Calif.,92038-2284, USA.) and according to the small scale rapid plasmid DNAisolation procedure described therein. The orientation of 5 of theresulting clones was checked using the restriction enzyme Clal. A clonewas chosen which gave the same pattern of restriction by ClaI as fd-tet,but which had no BstE II sites.

In vitro mutagenesis of fdTδBst was used to generate vectors havingappropriate restriction sites that facilitate cloning of antibodyfragments downstream of the gene III signal peptide and in frame withthe gene III coding sequence. The oligonucleotide directed mutagenesissystem version 2 (Amersham International) was used with oligo 1 (FIG. 4(i)) to create fdTPs/Bs (to facilitate cloning of VH fragments). Thesequence offdTPs/Bs (FIG. 4( i)) was confirmed using the sequenaseversion 2.0 kit (USB Corp., PO Box 22400, Cleveland, Ohio, 44122, USA.)with oligo 3 (FIG. 4) as a primer.

A second vector fdTPs/Xh (to facilitate cloning of single chain Fvfragments) was generated by mutagenising fdTPs/Bs with oligo 2 accordingto the method of Venkitaraman, A. R., Nucl. Acid Res. 17, p 3314. Thesequence of fdTPs/Xh (FIG. 4) was confirmed using the sequenase version2.0 kit (USB Corp.) with oligo 3 as a primer.

Clearly, alternative constructions will be apparent to those skilled inthe art. For example, M13 and/or its host bacteria could be modifiedsuch that its gene III could be disrupted without the onset of excessivecell death; the modified fd gene III, or other modified protein, couldbe incorporated into a plasmid containing a single stranded phagereplication origin, such as pUC119, superinfection with modified phagesuch as KO7 would then result in the encapsulation of the phage antibodygenome in a coat partially derived from the helper phage and partly fromthe phage antibody gene III construct.

The detailed construction of a vector such as fdTPs/Bs is only one wayof achieving the end of a phage antibody. For example, techniques suchas sticky feet cloning/mutagenesis (Clackson, T. and Winter, G. 1989Nucl. Acids. Res., 17, p 10163-10170) could be used to avoid use ofrestriction enzyme digests and/or ligation steps.

Example 2 Insertion of Immunoglobulin Fv Domain into Phage

The plasmid scFv D1.3 myc (gift from g. Winter and A. Griffiths)contains VH and VL sequences from the antibody D1.3 fused via a peptidelinker sequence to form a single chain Fv version of antibody D1.3. Thesequence of the scFv and surrounding sequences in scFvD1.3 myc is shownin FIG. 5.

The D1.3 antibody is directed against hen egg lysozyme (Harper, M. etal., 1987, Molec. Immunol. 24, 97-108) and the scFv form expressed in E.coli has the same specificity (A. Griffiths and G. Winter personalCommunication).

Digestion of scFv D1.3 myc with Pst1 and Xho1 (these restriction sitesare shown on FIG. 5), excises a fragment of 693 by which encodes thebulk of the scFv. Ligation of this fragment into fdTPs/Xh cleaved withPst1 and Xho1 gave rise to the construct fdTscFvD1.3 encoding the geneIII signal peptide and first amino acid fused to the complete D1.3 scFv,followed by the mature gene III protein from amino acid 2.

The vector fdTPs/Xh was prepared for ligation by digesting with the Pst1and Xho1 for 2 hours followed by digestion with calf intestinal alkalinephosphatase (Boehringer Mannheim UK Ltd., Bell Lane, Lewes, East Sussex,BN7 1LG) at one unit/ul for 30 minutes at 37° C. Fresh calf intestinalalkaline phosphatase was added to a final total concentration of 2units/ul and incubated for a further 30 minutes at 37° C. The reactionwas extracted three times with phenol/chloroform, precipitated withethanol and dissolved in water. The insert from scFvD1.3 myc was excisedwith the appropriate restriction enzymes (PstI and XhoI) extracted twicewith phenol/chloroform, precipitated with ethanol and dissolved inwater. Ligations were carried out as described in example 1, except bothvector and insert samples were at a final concentration of 5 ng/ul each.The formation of the correct construct was confirmed by sequencing asdescribed in example 1.

To demonstrate that proteins of the expected size were produced, virionswere concentrated by PEG precipitation as described above. The sampleswere prepared for electrophoresis as described in Sambrook J. et al 1989supra. The equivalent of 2 mls of supernatant was loaded onto an 18% SDSpolyacrylamide gel. After electrophoresis, the gel was soaked in gelrunning buffer (50 mM tris, 380 mM Glycine, 0.1% SDS) with 20% methanolfor 15 minutes. Transfer to nitrocellulose filter was executed in fresh1× running buffer/20% methanol using TE70 Semi Phor a semi-dry blottingapparatus (Hoeffer, 654 Minnesota Street, Box 77387, San Francisco,Calif. 94107, USA.).

After transfer, the filter was blocked by incubation for 1 hour in a 2%solution of milk powder (Marvel) in phosphate buffered saline (PBS).Detection of scFv and VH protein sequences in the phage antibody fusionproteins was effected by soaking the filter for 1 hour with a 1/1000dilution (in 2% milk powder) of a rabbit polyclonal antiserum raisedagainst affinity purified, bacterially expressed scFv fragment (giftfrom G. Winter). After washing with PBS (3×5 minute washes), boundprimary antibody was detected using an anti-rabbit antibody conjugatedto horseradish peroxidase (Sigma, Fancy Road, Poole, Dorset, BH17 7NH,UK.) for 1 hour. The filter was washed in PBS/0.1% triton X-100 anddeveloped with 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (DAB),0.02% cobalt chloride, 0.03% hydrogen peroxide in PBS.

The results showed that with clones fdTVHD1.3 (from example 3incorporating sequences coding for VH) and fdTscFvD1.3 (incorporatingsequences coding for scFv) a protein of between 69,000 and 92,500daltons is detected by the anti-Fv serum. This is the expected size forthe fusion proteins constructed. This product is not observed insupernatants derived from fd-tet, fdTδBst or fdTPs/Xh.

Example 3 Insertion of Immunoglobulin VH Domain into Phage Antibody

The VH fragment from D1.3 was generated from the plasmidpSW1-VHD1.3-TAG1 (Ward, E. S. et al., 1989 supra.). Digestion of thisplasmid with PstI and BstEII generates the fragment shown betweenpositions 113 and 432 in FIG. 5. Cloning of this fragment into the Pst1and BstEII sites of fdTPs/Bs gave rise to the construct fdTVHD1.3 whichencodes a fusion protein with a complete VH domain inserted between thefirst and third amino acids of the mature gene III protein (amino acidtwo has been deleted).

The methods used were exactly as in example 2 except that the vectorused was fdTPs/Bs digested with PstI and BstEII.

Example 4 Analysis of Binding Specificity of Phage Antibodies

The binding of the various phage antibodies to the specific antigen,lysozyme, was analysed using ELISA techniques. Phage antibodies (e.g.fdTVHD1.3 and fdTsc/FvD1.3) were grown in E. coli and Phage antibodyparticles were precipitated with PEG as described in the materials andmethods. Bound phage antibody particles were detected using polyclonalsheep serum raised against the closely related phage M13.

ELISA plates were prepared by coating 96 well plates (Falcon MicrotestIII flexible plate. Falcon: Becton Dickinson Labware, 1950 WilliamsDrive, Oxnard, Calif., 93030, USA.) with 200 ul of a solution oflysozyme (1 mg/ml unless otherwise stated) in 50 mm NaHCO₃ for 16-24hours. Before use, this solution was removed, the plate rinsed severaltimes in PBS and incubated with 200 ul of 2% milk powder/PBS for 1 hour.After rinsing several times with PBS, 100 ul of the test samples wereadded and incubated for 1 hour. Plates were washed (3 rinses in 0.05%Tween 20/PBS followed by 3 rinses in PBS alone). Bound phage antibodieswere detected by adding 200 ul/well of a 1/1000 dilution of sheepanti-M13 polyclonal antiserum (gift from G. Winter, although anequivalent antibody can be readily made by one skilled in the art usingstandard methodologies) in 2% milk powder/PBS and incubating for 1 hour.After washing as above, plates were incubated with biotinylatedanti-sheep antibody (Amersham International) for 30 minutes. Plates werewashed as above, and incubated with streptavidin-horseradish peroxidasecomplex (Amersham International). After a final wash as above, 0.5 mg/mlABTS substrate in citrate buffer was added (ABTS=2′2′-azinobis(3-ethylbenzothiazoline sulphonic acid); citrate buffer=50 mM citricacid, 50 mM tri-sodium citrate at a ratio of 54:46. Hydrogen peroxidewas added to a final concentration of 0.003% and the plates incubatedfor 1 hour. The optical density at 405 nm was read in a Titertekmultiskan plate reader.

FIG. 6 shows the effect of varying the amount of phage antibody. 100 ulof various dilutions of PEG precipitated phage were applied and theamount expressed in terms of the original culture volume from which itwas derived. Signals derived from both the scFv containing phageantibody (fdTscFvD1.3) and the VH containing phage antibody (fdTVHD1.3)and the VH containing phage antibody were higher than that derived fromthe phage antibody vector (fdTPs/Xh). The highest signal to noise ratiooccurs using the equivalent of 1.3 mls of culture.

FIG. 7 shows the results of coating the plates with varyingconcentrations of lysozyme or bovine serum albumin (BSA). The equivalentof 1 ml of the original phage antibody culture supernatant was used. Thesignals from supernatants derived from fdTscFvD1.3 were again higherthan those derived from fdTPs/Xh when lysozyme coated wells were used.There was no significant difference between these two types ofsupernatant when the plates were coated with BSA. Broadly speaking thelevel of signal on the plates is proportional to the amount of lysozymecoated. These results demonstrate that the binding detected is specificfor lysozyme as the antigen.

Example 5 Construction of fd CAT 2

It would be useful to design vectors that enable the use of restrictionenzymes that cut DNA infrequently, thus avoiding unwanted digestion ofthe antibody gene inserts within their coding sequence. Enzymes with aneight base recognition sequence are particularly useful in this respect,for example NotI and SfiI. Chaudhary et al (PNAS 87 p 1066-1070, 1990)have identified a number of restriction sites which occur rarely inantibody variable genes. The applicant has designed and constructed avector that utilises two of these sites, as an example of how this typeof enzyme can be used. Essentially sites for the enzymes ApaL1 and NotIwere engineered into fdTPs/Xh to create fdCAT2.

The oligonucleotide:

5′ACT TTC AAC AGT TTC TGC GGC CGC CCG TTT GAT CTC GAG CTC CTG CAG TTGGAC CTG TGC ACT GTG AGA ATA GAA 3′ (SEQ ID NO:4) was synthesised (supraFIG. 4 legend) and used to mutagenise fdTPs/Xh using an in vitromutagenesis kit from Amersham International as described in example 1,to create fd-CAT2. The sequence of fd-CAT2 was checked around the siteof manipulation by DNA sequencing. The final sequence around theinsertion point within gene III is shown in FIG. 8. N.B. fdCAT2 is alsoreferred to herein by the alternative terminologies fd-tet-DOG1 andfdDOG1.

Example 6 Specific Binding of Phage-Antibody (pAb) to Antigen

The binding of pAb D1.3 (fdTscFvD1.3 of example 2) to lysozyme wasfurther analysed by ELISA.

Methods.

1. Phage Growth.

Cultures of phage transduced bacteria were prepared in 10-100 mls 2×TYmedium with 15 μg/ml tetracycline and grown with shaking at 37° C. for16-24 hrs. Phage supernatant was prepared by centrifugation of theculture (10 min at 10,000 rpm, 8×50 ml rotor, Sorval RC-5B centrifuge).At this stage, the phage titre was 1-5×10₁₀/ml transducing units. Thephage were precipitated by adding ⅕ volume 20% PEG 2.5 M NaCl, leavingfor 1 hr at 4° C., and centrifuging (supra). The phage pellets wereresuspended in 10 mM Tris-HCl, 1 mM EDTA pH 8.0 to 1/100th of theoriginal volume, and residual bacteria and aggregated phage removed bycentrifugation for 2 min in a bench microcentrifuge.

ELISA

Plates were coated with antigen (1 mg/ml antigen) and blocked asdescribed in example 4. 2×10₁₀ phage transducing units were added to theantigen coated plates in phosphate buffered saline (PBS) containing 2%skimmed milk powder (MPBS). Plates were washed between each step withthree rinses of 0.5% Tween-20 in PBS followed by three rinses of PBS.Bound phage was developed by incubating with sheep anti-M13 antisera anddetected with horseradish peroxidase (HRP) conjugated anti-goat serum(Sigma, Poole, Dorset, UK) which also detects sheep immunoglobulins andABTS (2′2′-azinobis (3-ethylbenzothiazoline sulphonic acid). Readingswere taken at 405 nm after a suitable period. The results (FIG. 9) showthat the antibody bearing-phage had the same pattern of reactivity asthe original D1.3 antibody (Harper, M., Lema, F., Boulot, G., andPoljak, F. J. (1987) Molec. Immunol. 24, 97-108), and bound to henegg-white lysozyme, but not to turkey egg-white lysozyme, human lysozymeor bovine serum albumin. The specificity of the phage is particularlyillustrated by the lack of binding to the turkey egg-white lysozyme thatdiffers from hen egg-white lysozyme by only 7 amino acids.

Example 7 Expression of Fab D1.3

The aim of this example was to demonstrate that the scFv format used inexample 2 was only one way of displaying antibody fragments in the pAbsystem. A more commonly used antibody fragment is the Fab fragment(FIG. 1) and this example describes the construction of a pAb thatexpresses a Fab-like fragment on its surface and shows that it bindsspecifically to its antigen. The applicant chose to express the heavychain of the antibody fragment consisting of the VH and CH1 domains fromcoding sequences within the pAb itself and to co-express the light chainin the bacterial host cell infected with the pAb. The VH and CH1 regionsof ti-lysozyme antibody D1.3 were cloned in fd CAT2, and thecorresponding light chain cloned in plasmid pUC19. The work of Skerraand Pluckthun (Science 240, p 1038-1040 (1988) and Better et al 1988supra; demonstrated that multimeric antigen binding fragments of theantibody molecule could be secreted into the periplasm of the bacterialcell in a functional form using suitable signal sequences. However, inthese publications, special measures were described as being needed torecover the binding protein from the cell, for example Skerra andPluckham needed to recover the Fv fragment from the periplasm byaffinity chromatography. The present applicants have shown that it ispossible to direct the binding molecule to the outside of the cell on aphage particle, a process that requires several events to occur: correctsecretion and folding of the binding molecule; association of the chainsof the binding molecule; correct assembly of the phage particle; andexport of the intact phage particle from the cell.

Alternatively, it is possible however, to express the light chain fromwithin the pAb genome by, for example, cloning an expression cassetteinto a suitable place in the phage genome. Such a suitable place wouldbe the intergenic region which houses the multicloning sites engineeredinto derivative of the related phage M13 (see, for example,Yanisch-Perron, C. et al., Gene 33, p 103-119, (1985)).

The starting point for this example was the clone Fab D1.3 in pUC19, amap of which is shown in FIG. 10. The regions hybridising with theoligonucleotides KSJ6 and 7 below are shown underlined in FIG. 10. Thesequence encoding the VH-CH1 region (defined at the 5′ and 3′ edges bythe oligonucleotides KSJ6 and 7 below) was PCR amplified from Fab D1.3in pUC19 using oligonucleotides KSJ6 and 7, which retain the Pst I siteat the 5′ end and introduce a Xho I site at the 3′ end, to facilitatecloning into fd CAT2. The sequences for the oligonucleotides KSJ6 and 7are shown below. The underlined region of KSJ7 shows the portionhybridising with the sequence for D1.3. KSJ6: 5′ AGG TGC AGC TGC AGG AGTCAG G 3′ (SEQ ID NO:5)

KSJ7: 5′ GGT GAC CTC GAG TGA AGA TTT GGG CTC AAC TTT C 3′ (SEQ IDNO:6)-1

PCR conditions were as described in example II, except that thirtycycles of PCR amplification were performed with denaturation at 92° C.for 45 seconds, annealing at 55° C. for 1 minute and extension at 72° C.for 1 minute. The template used was DNA from TG1 cells containing FabD1.3 in pUC19 resuspended in water and boiled. The template DNA wasprepared from the colonies by picking some colony material into 100 μlof distilled H₂O and boiling for 10 mins. 1 μl of this mixture was usedin a 20 μl PCR. This regime resulted in amplification of the expectedfragment of approximately 600 bp. This fragment was cut with Pst I andXho I, purified from an agarose gel and ligated into Pst 1/Xho 1-cutfdCAT2. The PCR mixture was extracted with phenol/chloroform and ethanolprecipitated (Sambrook et al. supra.) before digestion with Pst1 andXho1 (New England Biolabs according to manufacturers recommendations.The fragment was resolved on 1% Tris-Acetate EDTA agarose gel (Sambrooket al. supra) and purified using Geneclean (BIO 101, Geneclean, LaJolla, San Diego, Calif., USA) according to manufacturersrecommendations.

fd-CAT2 vector DNA was digested with Pst 1 and Xho 1 (New EnglandBioLabs) according to manufacturers recommendations, extracted withphenol/chloroform and ethanol precipitated (Sambrook et al. supra.).

75 ng of Pst 1/Xho 1-digested vector DNA was ligated to 40 ng ofPCR-amplified PstI/Xho I-digested hEGF-R fragment in 12 μl of ligationbuffer (66 mM TrisHCl (pH7.6), 5 mM MgCl₂, 5 mM dithiothreitol, (100μg/ml bovine serum albumin, 0.5 mM ATP, 0.5 mM Spermidine) and 400 unitsT4 DNA ligase (New England BioLabs) for 16 hours at 16° C.

Two μl of the ligation mixture was transformed into 200 μl of competentE. coli MC1061 cells, plated on 2TY agar containing 15 μg/mltetracycline and incubated at 30° C. for 20 hours. A portion of theligation reaction mixture was transformed into E. coli MC1061 (Availablefrom, for example Clontech Laboratories Inc, Palo Alto, Calif.) andcolonies identified by hybridisation with the oligonucleotide D1.3CDR3Aas described in example 10. The presence of the VHCH1 gene fragment waslikewise confirmed by PCR, using oligonucleotides KSJ6 and 7. Arepresentative clone was called fd CAT2VHCH1 D1.3. The heavy chain wasdeleted from Fab D1.3 in pUC19 by Sph I cleavage of Fab D1.3 plasmidDNA. The pUC19 2.7 Kb fragment containing the light chain gene waspurified from a TAE agarose gel, and long of this DNA self-ligated andtransformed into competent E. coli TG1. Cells were plated on 2TY agarcontaining ampicillin (100 μg/ml) and incubated at 30° C. overnight. Theresulting colonies were used to make miniprep DNA (Sambrook et al.supra), and the absence of the heavy chain gene confirmed by digestionwith Sph I and Hind III. A representative clone was called LCD1.3 DHC.

An overnight culture of fd CAT2VHCH1 D1.3 cells was microcentrifuged at13,000×g for 10 minutes and 50 μl of the supernatant containing phageparticles added to 50 μl of an overnight culture of LCD1.3 DHC cells.The cells were incubated at 37° C. for 10 minutes and plated on 2TY agarcontaining ampicillin (100 μg/ml) and 15 μg/ml tetracycline. Phage wereprepared from some of the resulting colonies and assayed for theirability to bind lysozyme as described in example 6.

The results (FIG. 11) showed that when the heavy and light chain Fabderivatives from the original antibody D1.3 were present, the pAb boundto lysozyme. pAb expressing the fd VHCH1 fragment did not bind tolysozyme unless grown in cells also expressing the light chain. Thisshows that a functional Fab fragment was produced by an association ofthe free light chain with VHCH1 fragment fused to gene III and expressedon the surface of the pAb.

Example 8 Isolation of Specific, Desired Phage from a Mixture of VectorPhage

The applicant purified pAb (D1.3) (originally called fdTscFvD1.3 inexample 2) from mixtures using antigen affinity columns. pAb (D1.3) wasmixed with vector fd phage (see table 1) and approximately 10¹² phagepassed over a column of lysozyme-Sepharose (prepared from cyanogenbromide activated sepharose 4B (Pharmacia, Milton Keynes, Bucks, UK.)according to the manufacturers instructions. TG1 cells were infectedwith appropriate dilutions of the elutes and the colonies derived, wereanalysed by probing with an oligonucleotide that detects only the pAb(D1.3) see Table 1 and FIGS. 12A and 12B. A thousand fold enrichment ofpAb(D1.3) was seen with a single column pass. By growing the enrichedphage and passing it down the column again, enrichments of up to amillion fold were seen.

Enrichment was also demonstrated using purely immunological criteria.For example, 10¹² phage (at a ratio of 1 pAb (D1.3) to 4×10⁶ fdTPs/Bs)was subjected to two rounds of affinity selection, and then 26 coloniespicked and grown overnight. The phage was then assayed for lysozymebinding by ELISA (as example 6). Five colonies yielded phage withlysozyme binding activities, see table 1, and these were shown to encodethe scFv (D1.3) by PCR screening (example 13, using 30 cycles of 1minute at 92° C., 1 minute at 60° C., 1 minute at 72° C. using CDR3PCR1and oligo 3 (FIG. 4( i)) as primers).

Thus very rare pAbs can be fished out of large populations, by usingantigen to select and then screen the phage.

In this example, affinity chromatography of pAbs and oligonucleotideprobing were carried out as described below.

Approximately 10¹² phage particles in 1 ml MPBS were loaded onto a 1 mllysozyme-Sepharose affinity column which had been prewashed in MPBS. Thecolumn was washed in turn with 10 ml PBS; then 10 ml 50 mM Tris-HCl, 500mM Nacl pH 7.5; then 10 ml 50 mM Tris-HCl 500 mM NaCl pH 8.5; then 5 mls50 mM Tris-HCl, 500 mM NaCl pH 9.5 (adjusted with triethylamine) andthen eluted with 5 ml 100 mM triethylamine. The eluate was neutralisedwith 0.5 M sodium phosphate buffer pH 6.8 and the phage plated foranalysis. For a second round of affinity chromatography, the firstcolumn eluate was plated to about 30,000 colonies per petri dish. Afterovernight growth, colonies were then scraped into 5 ml 2×TY medium, anda 20 μl aliquot diluted into 10 ml fresh medium and grown overnight. Thephage was PEG precipitated as described above, resuspended in 1 ml MPBSand loaded onto the column, washed and eluted as above.

Oligonucleotides Sythesised:

CDR3PCR1 5′TGA GGA C(A or T) C(A or T) GC CGT CTA CTA CTG TGC 3′ (SEQ IDNO:7)

40 pmole of oligonucleotide VH1FOR (Ward, E. S., et al (1989) Nature341, 544-546), specific to pAb (D1.3) was phosphorylated with 100 μCiα-32P ATP, hybridised (1 pmole/ml) to nitrocellulose filters at 67° C.in 6× saline sodium citrate (SSC) Sambrook et al., supra. buffer for 30minutes and allowed to cool to room temperature for 30 mins, washed 3×1min at 60° C. in 0.1×SSC.

Example 9 Construction of pAb Expressing Anti-Hapten Activity

Oxazolone is a hapten that is commonly used for studying the details ofthe immune response. The anti-oxazalone antibody, NQ11 has beendescribed previously (E. Gherardi, R. Pannell, C. Milstein, J. Immunol.Method 126 61-68). A plasmid containing the VH and VL gene of NQ11 wasconverted to a scFv form by inserting the BstEII/SacI fragment ofscFvD1.3 myc (nucleotides 432-499 of FIG. 5) between the VH and VL genesto generate pscFvNQ11, the sequence of which is shown in FIG. 13. ThisscFv was cloned into the Pst1/Xho1 site of FdTPs/Xh (as describedearlier) to generate pAb NQ11 has an internal Pst1 site and so it wasnecessary to do a complete digest of pscFvNQ11 with Xho1 followed by apartial digest with Pst1).

The specific binding of pAb NQ11 was confirmed using ELISA. ELISA plateswere coated at 37° C. in 50 mM NaHCO₃ at a protein concentration of 200mg/ml. Plates were coated with either hen egg lysozyme (HEL), bovineserum albumin (BSA), or BSA conjugated to oxazolone (OX-BSA) (method ofconjugation in Makela O., Kartinen M., Pelkonen J. L. T., Karjalainen K.(1978) J. Exp. Med. 148 1644). Preparation of phage, binding to ELISAplates, washing and detection was as described in example 6. Sampleswere assayed in duplicate and the average absorbance after 10 minutespresented in FIG. 14.

This result demonstrates that the pAb NQ11 binds the correct antigen.FIG. 14 also shows that pAb D1.3 and pAb NQ11 bind only to the antigenagainst which the original antibodies were raised.

Example 10 Enrichment of pAb D1.3 from Mixtures of Other pAb by AffinityPurification

3×10¹⁰ phage in 10 mls of PBSM at the ratios of pAb D1.3 to pAb NQ11shown in table 2 were passed over a 1 ml lysozyme Sepharose column.Washing, elution and other methods were as described in example 8 unlessotherwise stated. Eluates from the columns were used to infect TG1 cellswhich were then plated out. Colonies were probed with a probe whichdistinguishes pAb D1.3 from pAb NQ11. The sequence of thisoligonucleotide (D1.3CDR3A) is:—

5′GTA GTC AAG CCT ATA ATC TCT CTC 3′ (SEQ ID NO:8). Table 2 presents thedata from this experiment. An enrichment of almost 1000 fold wasachieved in one round and an enrichment of over a million fold in tworounds of purification. This parallels the result described in example8.

Example 11 Insertion of a Gene Encoding an Enzyme (Alkaline Phosphatase)into fd-CAT2

As an example of the expression of a functional enzyme on thebacteriophage surface, the applicants have chosen bacterial alkalinephosphatase, an enzyme that normally functions as a dimer (McCracken, S,and Meighen, E., J. Biol. Chem. 255, p 2396-2404, (1980)). Theoligonucleotides were designed to generate a PCR product with an Apa L1site at the 5′ end of phoA gene and a Not 1 site at its 3′ end, thusfacilitating cloning into fd-CAT 2 to create a gene III fusion protein.The oligonucleotides synthesised were: phoA 1:5′ TAT TCT CAC AGT GCA CAAACT GTT GAA CGG ACA CCA GAA ATG CCT GTT CTG 3′ (SEQ ID NO:9) and,phoA2:5′ ACA TGT ACA TGC GGC CGC TTT CAG CCC CAG AGC GGC TTT C 3′ (SEQID NO:10). The sequence of the phoA gene is presented in Chang C. N. etal., Gene 44, p 121-125 (1986). The plasmid amplified (pEK86) containsan alkaline phosphate gene which differs from the sequence of Chang etal, by a mutation which converts arginine to alamine at position 166.

The PCR reaction was carried out in 100 μl of 10 mM Tris/HCl pH 8.3,containing 50 mM KCl, 5 mMdNTP 2.5 mM MgCl₂, 0.01% gelatin, 0.25units/μl of Taq polymerase (Cetus/Perkin Elmer) and 0.5 μg/ml template.The template was the pEK86 plasmid (described by Chaidaroglou et al.,Biochemistry 27 p 8338-8343, 1988). The PCR was carried out in a Techne(Techne, Duxford, Cambridge, UK) PHC-2 dri-block using thirty cycles of1 min at 92° C., 2 min at 50° C., 3 min at 72° C.

The resultant product was extracted with phenol:chloroform, precipitatedwith ethanol, and the pellet dissolved in 35 μl water. Digestion with0.3 units/μl of Apa L1 was carried out in 150 μl volume according tomanufacturers instructions for two hours at 37° C. After heatinactivation of the enzyme at 65° C., NaCl was added to a finalconcentration of 150 mM and 0.4 units/μl, Not1 enzyme added. Afterincubation for 2 hours at 37° C., the digest was extracted withphenol:chloroform and precipitated as above, before being dissolved in30 μl of water. The vector fd-CAT2 was sequentially digested with Apa L1and Not1 according to the manufacturers instructions and treated withcalf intestinal alkaline phosphatase as described in example 2. Thesample was extracted three times with phenol:chloroform, precipitatedwith ethanol and dissolved in water. The ligations were performed with afinal DNA concentration of 1-2 ng/μl of both the cut fd-CAT2 and thedigested PCR product. The ligations were transformed into competent TG1cells and plated on 2×TY tet plates. Identification of clones containingthe desired insert was by analytical PCR performed using the conditionsand primers above, on boiled samples of the resulting colonies. Thecorrect clone containing the phoA gene fused in frame to gene III wascalled fd-phoAla 166. The sequence at the junction of the cloning regionis given in FIG. 15.

Example 12 Measuring Enzyme Activity of Phage-Enzyme

Overnight cultures of TG1 or KS272 (E. coli cells lacking phoA. StrauchK. L., and Beckwith J. PNAS 85 1576-1580, 1988) cells containing eitherfd-phoAla 166 or fd-CAT2 were grown at 37° C. in 2×TY with 15 μg/mltetracycline. Concentrated, PEG precipitated phage were prepared asdescribed earlier. Enzyme assays (Malamy, M. H. and Horecker B. L.,Biochemistry 3, p 1893-1897, (1964)) were carried out at 24° C. in afinal concentration of 1M Tris/HCl pH 8.0, 1 mM 4-nitrophenyl phosphate(Sigma), 1 mM MgCl₂. 100 μl of a two times concentrate of this reactionmixture was mixed with 100 μl of the test sample in a 96 well plate.Absorbance readings were taken every minute for 30 minutes at awavelength of 405 nm in a Titretek Mk 2 plate reader. Initial reactionrates were calculated from the rate of change of absorbance using amolar absorbance of 17000 l/mol/cm.

Standard curves (amount of enzyme vs. rate of change of absorbance) wereprepared using dilutions of purified bacterial alkaline phosphatase(Sigma type III) in 10 mM Tris/HCl pH 8.0, 1 mM EDTA. The number ofenzyme molecules in the phage samples were estimated from the actualrates of change of absorbance of the phage samples and comparison tothis standard curve.

The results in Table 3 show that alkaline phosphatase activity wasdetected in PEG precipitated material in the sample containingfd-phoAla166 but not fd-CAT2. Furthermore, the level of activity wasconsistent with the expected number of 1-2 dimer molecules of enzyme perphage. The level of enzyme activity detected was not dependent on thehost used for growth. In particular, fd-phoA1a166 grown on phoA minushosts showed alkaline phosphatase activity.

Therefore, the phage expressed active alkaline phosphatase enzyme, fromthe phoA-gene III fusion, on the phage surface.

Example 13 Insertion of Binding Molecules into Alternative Sites in thePhage

The availability of an alternative site in the phage for the insertionof binding molecules would open up the possibility of more easilyexpressing more than one binding molecule e.g. an antibody fragment in asingle pAb. This may be used to generate single or multiple bindingspecificities. The presence of two distinct binding activities on asingle molecule will greatly increase the utility and specificity ofthis molecule. It may be useful in the binding of viruses with a highmutational rate such as human immunodeficiency virus. In addition, itmay be used to bring antigens into close proximity (e.g. drug targetingor cell fusion) or it may act as a “molecular clamp” in chemical,immunological or enzymatic processes.

The vector fd-tet and the derivatives described here, have a singleBamH1 site in gene 3. This has previously been used for the expressionof peptide fragments on the surface of filamentous bacteriophage (SmithG P. (1985) Science 228 p 1315-1317 and de la Cruz et al. (1988) J.Biol. Chem. 263 p 4318-4322). This provides a potential alternative sitefor the insertion of antibody fragments.

DNA fragments encoding scFv's from D1.3 or NQ11 were generated by PCRusing the primers shown below. These primers were designed to generate afragment with BamH1 sites near both the termini, to enable cloning intothe BamH1 site of gene3 (see FIG. 16( i)). The oligonucleotides used,also ensure that the resulting PCR product lacks Pst1 and Xho1restriction sites normally used for manipulating the scFv's (see FIG.16(1)). This will facilitate subsequent manipulation of a secondantibody fragment in the usual way at the N terminus of gene 3. Theoligonucleotides used were:—

G3Bam1 5′TTT AAT GAG GAT CCA CAG GTG CAG CTG CAA GAG 3′ (SEQ ID NO:11)

G3Bam2 5′AAC GAA TGG ATC CCG TTT GAT CTC AAG CTT 3′ (SEQ ID NO:12).

Preparation of Vector and PCR Insert

The PCR reaction was carried out in an 80 μl reaction as described inexample 11 using 1 ng/μl of template and 0.25 U/μl of Tag polymerase anda cycle regime of 94° C. for 1 minute, 60° C. for 1 minute and 70° C.for 2 minutes over 30 cycles. The template was either pscFvNQ11 (example9) or scFvD1.3 myc (example 2). Reaction products were extracted withphenol:chloroform, precipitated, dissolved in water and digested withBamH1 according to manufacturers instructions. The digest wasre-extracted with phenol: chloroform, precipitated and dissolved inwater.

The vector fdTPs/Xh was cleaved with BamH1 and treated with calfintestinal phosphatase and purified as described in example 2. Ligationswere set up at a vector concentration of approximately 6 ng/μl and a PCRinsert concentration of approximately 3 ng/μl. These were ligated for2.5 hours at room temperature before transforming into competent TG1cells and plating on TY tet plates. The resultant colonies were probedas described in example 8. DNA was prepared from a number of coloniesand the correct orientation and insert size confirmed by restrictiondigestion with Hind III in isolation or in combination with BamH1. (OneHind III site is contributed by one of the primers and the other by thevector).

Two clones containing a D1.3 insert (fdTBam1) and fdTBam2) and onecontaining an NQ11 insert (NQ11Bam1) were grown up and phage prepared asdescribed earlier. ELISAs were carried out as described in example 6. Nospecific signal was found for any of these clones suggesting that thenatural BamHI site is not a suitable site for insertion of a functionalantibody (results not shown).

It may be possible to clone into alternative sites to retain bindingactivity. The peptide repeats present in gene III may provide such asite (FIG. 16 blocks A and B). This can be done by inserting a BamH1site and using the PCR product described above. To facilitate this, thenatural BamH1 site was removed by mutagenesis with the oligonucleotideG3mutδBam shown below (using an in vitro mutagenesis kit (AmershamInternational)):—

G3mutδBam 5′ CA AAC GAA TGG GTC CTC CTC ATT A 3′ (SEQ ID NO:13)

The underlined residue replaces an A residue, thereby removing the BamH1site. DNA was prepared from a number of clones and several mutantslacking BamH1 sites identified by restriction digestion.

The oligonucleotide G3 Bamlink was designed to introduce a BamH1 site ata number of possible sites within the peptide linker sites A and B, seeFIG. 16( ii). The sequence of the linker is: Bamlink 5′CC (G or A) CCACC CTC GGA TCC (G or A) CC ACC CTC 3′ (SEQ ID NO:14). Its relationshipto the peptide repeats in gene III is shown in FIG. 16.

Example 14 PCR Assembly of Mouse VH and VL Kappa (VLK) Repertoires forPhage Display

The principle is illustrated in FIG. 17. Details are provided insections A to F below but the broad outline is first discussed.

-   1. cDNA is prepared from spleen RNA from an appropriate mouse and    the VH and VLK repertories individually amplified. Separately,    primers reverse and complementary to VH1FOR-2 (domain 1) and    VLK2BACK (domain 2) are used to amplify an existing scFv-containing    DNA by PCR. (The term FOR refers to e.g. a primer for amplification    of sequences on the sense strand resulting in antisense coding    sequences. The term BACK refers to e.g. a primer for amplification    of sequences on the antisense strand resulting in sense coding    sequences). This generates a ‘linker’ molecule encoding the linker    with the amino acid sequence (1 letter code) (GGGGS)₃ (SEQ ID NO:15)    which overlaps the two primary (VH and VLK) PCR products.-   2. The separate amplified VH, VLK and linker sequences now have to    be assembled into a continuous DNA molecule by use of an ‘assembly’    PCR. In the secondary ‘assembly’ PCR, the VH, VLK and linker bands    are combined and assembled by virtue of the above referred to    overlaps. This generates an assembled DNA fragment that will direct    the expression of VH and one VLK domain. The specific VH/VLK    combination is derived randomly from the separate VH and VLK    repertoires referred to above.    -   The assembly PCR is carried out in two stages. Firstly, 7 rounds        of cycling with just the three bands present in the PCR,        followed by a further 20 rounds in the presence of the flanking        primers VH1BACK (referring to domain 1 of VH) and VLKFOR. The        nucleotide sequences for these oligonucleotide primers are        provided under the section entitled ‘Primer Sequences’ below.        This two stage process, avoids the potential problem of        preferential amplification of the first combinations to be        assembled.

For cloning into the phage system, the assembled repertoires must be‘tagged’ with the appropriate restriction sites. In the example providedbelow this is illustrated by providing an ApaL1 restriction site at theVH end of the continuous DNA molecule and a Not 1 site at the VLK end ofthe molecule. This is carried out by a third stage PCR using taggedprimers. The nucleotide sequences for these oligonucleotide primers arealso provided under the section entitled ‘Primer Sequences’ below. Thereare however, 4 possible kappa light chain sequences (whereas a singleconsensus heavy chain sequence can be used). Therefore 4 oligonucleotideprimer sequences are provided for VLK.

For this third stage PCR, sets of primers which create the newrestriction site and have a further 10 nucleotides on the 5′ side of therestriction site have been used. However, long tags may give bettercutting, in which case 15-20 nucleotide overhangs could be used.

Scrupulously clean procedures must be used at all times to avoidcontamination during PCR. Negative controls containing no DNA mustalways be included to monitor for contamination. Gel boxes must bedepurinated. A dedicated Geneclean kit (B10 101, Geneclean, La Jolla,San Diego, Calif., USA) can be used according to manufacturersinstructions to extract DNA from an agarose gel. The beads, NaI and theNEW wash should be aliquoted.

All enzymes were obtained from CP Laboratories, P.O. Box 22, Bishop'sStortford, Herts CM20 3DH and the manufacturers recommended and suppliedbuffers were used unless otherwise stated.

A. RNA Preparation

RNA can be prepared using may procedures well known to those skilled inthe art. As an example, the following protocol (Triton X-100 lysis,phenol/SDS RNase inactivation) gives excellent results with spleen andhybridoma cells (the addition of VRC (veronal ribosyl complex) as anRNase inhibitor is necessary for spleen cells). Guanidiniumisothiocyanate/CsC1 procedures (yielding total cellular RNA) also givegood results but are more time-consuming.

-   1. Harvest 1 to 5×10⁷ cells by centrifugation in a bench tope    centrifuge at 800×g for 10 minutes at 4° C. Resuspend gently in 50    ml of cold PBS buffer. Centrifuge the cells again at 800×g for 10    minutes at 4° C., and discard supernatant.-   2. On ice, add 1 ml ice-cold lysis buffer to the pellet and    resuspend it with a 1 ml Gilson pipette by gently pipetting up and    down. Leave on ice for 5 minutes.-   3. After lysis, remove cell debris by centrifuging at 1300 rpm for 5    minutes in a microfuge at 4° C., in precooled tubes.-   4. Transfer 0.5 ml of the supernatant to each of two eppendorfs    containing 60 μl 10% (w/v) SDS and 250 μl phenol (previously    equilibrated with 100 mM Tris-HCl pH 8.0). Vortex hard for 2    minutes, then microfuge (13000 rpm) for five minutes at room    temperature. Transfer the upper, aqueous, phase to a fresh tube.-   5. Re-extract the aqueous upper phase five times with 0.5 ml of    phenol.-   6. Precipitate with 1/10 volume 3M sodium acetate and 2.5 volumes    ethanol at 20° C. overnight or dry ice-isopropanol for 30 minutes.-   7. Wash the RNA pellet and resuspended in 50 μl to check    concentration by OD260 and check 2 μg on a 1% agarose gel. 40 μg of    RNA was obtained from spleen cells derived from mice.    Lysis buffer is [10 mM Tris-HCl pH 7.4, 1 mM MgCl2, 150 mM NaCl, 10    mM VRC (New England Biolabs), 0.5% (w/v) Triton X-100], prepared    fresh.

Lysis buffer is [10 mM Tris-HCl pH 7.4, 1 mM MgCl₂, 150 mM NaCl, 10 mMVRC (New England Biolabs), 0.5% (w/v) Triton X-100], prepared fresh.

B. cDNA Preparation

cDNA can be prepared using many procedures well known to those skilledin the art. As an example, the following protocol can be used:

-   1. Set up the following reverse transcription mix:

μl H₂O (DEPC-treated) 20 5 mM dNTP 10 10 × first strand buffer 10 0.1 MDTT 10 FOR primer(s) (10 pmol/μl) 2 (each) (see below) RNasin (Promega;40 U/μl)  4NB

-   i) DEPC is diethylpyrocarbonate, the function of which is to    inactivate any enzymes that could degrade DNA or RNA-   ii) dNTP is deoxynucleotide triphosphate-   iii) DTT is dithiothreitol the function of which is as an    antioxidant to create the reducing environment necessary for enzyme    function.-   iv) RNasin is a ribonuclease inhibitor obtained from Promega    Corporation, 2800 Woods Hollow Road, Madison, Wis., USA.-   2. Dilute 10 μg RNA to 40 μl final volume with DEPC-treated water.    Heat at 65° C. for 3 minutes and hold on ice for one minute (to    remove secondary structure).-   3. Add to the RNA the reverse transcription mix (58 μl) and 4 μl of    the cloned reverse transcriptase ‘Super RT’ (Anglian Biotech Ltd.,    Whitehall House, Whitehall Road, Colchester, Essex) and incubate at    42° C. for one hour.-   4. Boil the reaction mix for three minutes, cool on ice for one    minute and then spin in a microfuge to pellet debris. Transfer the    supernatant to a new tube.

10× first strand buffer is [1.4M KCl, 0.5M Tris-HCl pH 8.1 at 42° C. 80mM MgCl₂].

The primers anneal to the 3′ end. Examples of kappa light chain primersare MJK1FONX, MJK2FONX, MJK4FONX and MJK5FONX (provided under ‘PrimerSequences’ below) and examples of heavy chain primers are MIGG1, 2 (CTGGAC AGG GAT CCA GAG TTC CA) (SEQ ID NO:16) and MIGG3 (CTG GAC AGG GCTCCA TAG TTC CA) (SEQ ID NO:17) which anneal to CH1.

Alternatively, any primer that binds to the 3′ end of the variableregions VH, VLK, VL, or to the constant regions CH₁, CK or CL can beused.

C. Primary PCRs

For each PCR and negative control, the following reactions are set up(e.g. one reaction for each of the four VLKs and four VH PCRs). In thefollowing, the Vent DNA polymerase sold by (C.P. Laboratories Ltd (NewEngland Biolabs) address given above) was used. The buffers are asprovided by C.P. Laboratories.

μl H₂O 32.5 10 × Vent buffer 5 20 × Vent BSA 2.5 5 mM dNTPs 1.5 FORprimer 10 pmol/μl) 2.5 BACK primer 10 pmol/μl 2.5The FOR and BACK primers are given in the section below entitled ‘PrimerSequences’. For VH, the FOR primer is VH1FOR-2 and the BACK primer isVH1BACK. For VLK the FOR primers are MJK1FONX, MJK2FONX, MJK4FONX andMJK5FONX (for the four respective kappa light chains) and the BACKprimer is VK2BACK. Only one kappa light chain BACK primer is necessary,because binding is to a nucleotide sequence common to the four kappalight chains.

UV this mix 5 minutes. Add 2.5 μl cDNA preparation (from B above), 2drops paraffin oil (Sigma Chemicals, Poole, Dorset, UK). Place on acycling heating block, e.g. PHC-2 manufactured by Techne Ltd. DuxfordUK, pre-set at 94° C. Add 1 μl Vent DNA polymerase under the paraffin.Amplify using 25 cycles of 94° C. 1 min, 72° C. 2 min. Post-treat at 60°C. for 5 min.

Purify on a 2% lmp (low melting point agarose/TAE (tris-acetate EDTA)gel and extract the DNA to 20 μl H₂O per original PCR using a GENECLEANkit (see earlier; Bio 101, La Jolla Calif., USA) in accordance with themanufacturers instructions.

D. Preparation of Linker

Set up in bulk (e.g. 10 times)

μl H₂O 34.3 10 × Vent buffer 5 20 × Vent BSA 2.5 5 mM dNTPs 2 LINKFORprimer 10 pmol/μl) 2.5 LINKBACK primer 10 pmol/μl 2.5 DNA from fcFv D1.3(example 2) 1 Vent enzyme 0.2The FOR and BACK primers are given in the section below entitled ‘PrimerSequences’. The FOR primer is LINKFOR and the BACK primer is LINKBACK.Cover with paraffin and place on the cycling heating block (see above)at 94° C. Amplify using 25 cycles of 94° C. 1 min, 65° C. 1 min, 72° C.2 min. Post-treat at 60° C. for 5 min.

Purify on 2% lmp/TAE gel (using a loading dye without bromophenol blueas a 93 bp fragment is desired) and elute with SPIN-X column (CostarLimited, 205 Broadway, Cambridge, Ma. USA,) and precipitation. Take upin 5 μl H₂O per PCR reaction.

E. Assembly PCRs

A quarter of each PCR reaction product (5 μl) is used for each assembly.The total volume is 25 μl.

For each of the four VLK primers, the following are set up:

H₂O 4.95 10 × Vent buffer 2.5 20 × Vent BSA 1.25 5 mM dNTPs 0.8UV irradiate this mix for 5 min. Add 5 μl each of Vh andVK band from the primary PCRs and 1.5 μl of linker as isolated from thepreparative gels and extracted using the Geneclean kit as described in Cand D above. Cover with paraffin. Place on the cycling heating blockpreset at 94° C. Add 1 μl Vent under the paraffin. Amplify using 7cycles of 94° C. 2 min, 72° C. 4 min. Then return the temperature to 94°C.

Add 1.5 μl each of VH1BACK and the appropriate VKFOR primers MJK1FONX,MJK2FONX, MJK4FONX or MJK5FONX (10 pmol/μl) at 94° C. The primers shouldhave been UV-treated as above. Amplify using 20 cycles of 94° C. 1.5min, 72° C. 2.5 min. Post-treat at 60° C. for 5 min. Purify on 2%lmp/TAE gel and extract the DNA to 20 μl H₂O per assembly PCR using aGeneclean kit (see earlier) in accordance with the manufacturersinstructions.

F. Adding Restriction Sites

For each assembly and control set up:

μl H₂O 36.5 10 × Taq buffer 5 5 mM dNTPs 2 FOR primer (10 pmol/μl) 2.5BACK primer (10 pmol/μl) 2.5 Assembly product 1The FOR and BACK primers are given in the section below entitled ‘PrimerSequences’. The FOR primer is any of JK1NOT10, JK2NOT10, JK4NOT10 orJK5NOT10 (for the four respective kappa light chains) for putting a NotIrestriction site at the VLK end. The BACK primer is HBKAPA10 for puttingan ApaL1 restriction site at the VH end.

Cover with paraffin and place on the cycling heating block preset at 94°C. Add 0.5 μl Cetus Taq DNA polymerase (Cetus/perkin-Elmer,Beaconsfield, Bucks, UK) under the paraffin. Amplification is carriedout using 11 to 15 rounds of cycling (depends on efficiency) at 94° C. 1min, 55° C. 1 min, 72° C. 2 min. Post-treat at 60° C. for 5 min.

10×Taq buffer is [0.1M Tris-HCl pH 8.3 at 25° C., 0.5M KCl, 15 mM MgCl₂,1 mg/ml gelatin].

G. Work-Up

Purify once with CHCl₃/IAA (isoamylalcohol), once with phenol, once withCHCl₃/IAA and back-extract everything to ensure minimal losses.Precipitate and wash twice in 70% EtOH. Dissolve in 70 μl H₂O.

Digest overnight at 37° C. with NotI: μl

μl DNA (joined seq) 70 NEB NotI buffer × 10 10 NEB BSA × 10 10 Not1 (10U/μl) 10The DNA (joined sequence) above refers to the assembled DNA sequencecomprising in the 5′ to 3′ direction

ApaL1 restriction site

VH sequence

Linker sequence

VLK sequence

Not 1 restriction site.

The VLK sequence may be any one of four possible kappa chain sequences.

The enzymes Not 1 above, ApaL1 below and the buffers NEB Not 1, NEB BSAabove and the NEB buffer 4 (below) are obtainable from CP Laboratories,New England Biolabs mentioned above.

Re-precipitate, take up in 80 μl H₂O. Add to this 10 μl NEB buffer 4 and10 μl Apal 1.

Add the enzyme ApaL1 in aliquots throughout the day, as it has a shorthalf-life at 37° C.

Purify on 2% lmp/TAE gel and extract the DNA using a Geneclean kit, inaccordance with the manufacturers instructions. Redigest if desired.

H. Final DNA product

The final DNA product is an approximate 700 by fragment with Apa L1 andNot1 compatible ends consisting of randomly associated heavy and lightchain sequences linked by a linker. A typical molecule of this type isthe scFvD1.3 molecule incorporated into fdscFvD1.3 described in example3. These molecules can then be ligated into suitable fd derived vectors,e.g. fdCAT2 (example 5), using standard techniques.

Primer Sequences

Primary PCR oligos (restrictions sites underlined):

VH1FOR-2 TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC (SEQ ID NO:18)

VH1BACK AGG TSM ARC TGC AGS AGT CWG G (SEQ ID NO:19)

MJK1FONX CCG TTT GAT TTC CAG CTT GGT GCC (SEQ ID NO:20)

MJK2FONX CCG TTT TAT TTC CAG CTT GGT CCC (SEQ ID NO:21)

MJK4FONX CCG TTT TAT TTC CAA CTT TGT CCC (SEQ ID NO: 22)

MJK5FONX CCG TTT CAG CTC CAG CTT GGT CCC (SEQ ID NO:23)

VK2BACK GAC ATT GAG CTC ACC CAG TCT CCA (SEQ ID NO:24)

Ambiguity codes M=A or C, R=A or G, S=G or C,

W=A or T

PCR oligos to make linker:

LINKFOR TGG AGA CTC GGT GAG CTC AAT GTC (SEQ ID NO:25)

LINKBACK GGG ACC ACG GTC ACC GTC TCC TCA (SEQ ID NO:26)

For adding restriction sites:

-   HBKAPA10 CAT GAC CAC AGT GCA CAG GTS MAR CTG CAG SAG TCW GG (SEQ ID    NO:27)-   JKINOT10 GAG TCA TTC TGC GGC CGC CCG TTT GAT TTC CAG CTT GGT GCC    (SEQ ID NO:28)-   JK2NOT10 GAG TCA TTC TGC GGC CGC CCG TTT TAT TTC CAG CTT GGT CCC    (SEQ ID NO:29)-   JK4NOT10 GAG TCA TTC TGC GGC CGC CCG TTT TAT TTC CAA CTT TGT CCC    (SEQ ID NO:30)-   JK5NOT10 GAG TCA TTC TGC GGC CGC CCG TTT CAG CTC CAG CTT GGT CCC    (SEQ ID NO:31)

Example 15 Insertion of the Extracellular Domain of a Human Receptor forPlatelet Derived Growth Factor (PDGF) Isoform BB into fd CAT2

A gene fragment encoding the extracellular domain of the human receptorfor platelet derived growth factor isoform BB (h-PDGFB-R) was isolatedby amplification, using the polymerase chain reaction, of plasmid RP41,(from the American Type Culture collection, Cat. No. 50735), a cDNAclone encoding amino-acids 43 to 925 of the PDGF-B receptor (Gronwald,R. G. K. et al PNAS 85 p 3435-3439 (1988)). Amino acids 1 to 32 ofh-PDGFB-R constitute the signal peptide. The oligonucleotide primerswere designed to amplify the region of the h-PDGFB-R gene correspondingto amino acids 43 to 531 of the encoded protein. The primer RPDGF3 forthe N-terminal region also included bases encoding amino acids 33 to 42of the h-PDGFB-R protein (corresponding to the first ten amino acidsfrom the N-terminus of the mature protein) to enable expression of thecomplete extracellular domain. The primers also incorporate a uniqueApaL1 site at the N-terminal end of the fragment and a unique Xho1 siteat the C terminal end to facilitate cloning into the vector fdCAT2. Thesequence of the primers is:

-   RPDGF3 5′ CAC AGT GCA CTG GTC GTC ACA CCC CCG GGG CCA GAG CTT GTC    CTC AAT GTC TCC AGC ACC TTC GTT CTG 3′ (SEQ ID NO:32)-   RPDGF2 5′ GAT CTC GAG CTT AAA GGG CAA GGA GTG TGG CAC 3′ (SEQ ID    NO:33)

PCR amplification was performed using high fidelity conditions (Eckert,K. A. and Kunkel, T. A. 1990 Nucl Acids Research 18 3739-3744). The PCRmixture contained: 20 mM TrisHCl (pH7.3 at 70° C., 50 mM KCl, 4 mMmagnesium chloride, 0.01% gelatin, 1 mM each of dATP, dCTP, dGTP anddTTP, 500 ng/ml RP41 DNA, each primer and 50 units/ml Taq polymerase(Cetus/Perkin Elmer, Beaconsfield, Bucks, U.K.). Thirty cycles of PCRwere performed with denaturation at 92° C. for 1 min, annealing at 60°C. for 1 min and extension at 72° C. for 1.5 min. This reaction resultedin amplification of a fragment of ca. 1500 bp as expected.

fdCAT2 vector DNA (see example 5) was digested with ApaL1 and Xho1 (NewEngland Biolabs) according to manufacturers recommendations, extractedwith phenol/chloroform and ethanol precipitated (Sambrook et al, supra).Cloning of amplified RP41 DNA into this vector and identification of thedesired clones was performed essentially as in example 7 except thatdigestion of the PCR product was with ApaL1 and Xho 1. Coloniescontaining h-PDGFB-R DNA were identified by probing with 32p labelledRPDGF2 and the presence of an insert in hybridising colonies wasconfirmed by analytical PCR using RPDGF3 and RPDGF2 using the conditionsdescribed in example 7.

Example 16 Binding of 125I-PDGF-BB to the Extracellular Domain of theHuman Receptor for Platelet Derived Growth Factor Isoform BB Displayedon the Surface of fd Phage. Measured using an Immunoprecipitation Assay

Phage particles, expressing the extracellular domain of the humanplatelet derived growth factor isoform BB receptor (fd h-PDGFB-R), wereprepared by growing E. coli MC1061 cells transformed with fd h-PDGFB-Rin 50 ml of 2×TY medium with 15 ug/ml tetracycline for 16 to 20 hours.Phage particles were concentrated using polyethylene glycol as describedin example 6 and resuspended in PDGF binding buffer (25 mM HEPES, pH7.4,0.15 mM NaCl, 1 mM magnesium chloride, 0.25% BSA) to 1/33rd of theoriginal volume. Residual bacteria and undissolved material were removedby spinning for 2 min in a microcentrifuge. Immunoblots using anantiserum raised against gene III protein (Prof. I. Rashed, Konstanz,Germany) show the presence in such phage preparations of ageneIII-h-PDGFB-R protein of molecular mass 125000 corresponding to afusion between h-PDGFB-R external domain (55000 daltons) and geneIII(apparent molecular mass 70000 on SDS-polyacrylamide gel).

Duplicate samples of 35 μl concentrated phage were incubated with¹²⁵I-PDGF-BB (78.7 fmol, 70 nCi, 882 Ci/mmol; Amersham Internationalplc, Amersham, Bucks) for 1 hour at 37° C. Controls were included inwhich fdTPs/Bs vector phage (FIG. 4) or no phage replaced fd h-BDGFB-Rphage. After this incubation, 10 ul of sheep anti-M13 polyclonalantiserum (a gift from M. Hobart) was added and incubation continued for30 min at 20° C. To each sample, 40 ul (20 ul packed volume) of proteinG Sepharose Fast Flow (Pharmacia, Milton Keynes) equilibrated in PDGFbinding buffer was added. Incubation was continued for 30 min at 20° C.with mixing by end over end inversion on a rotating mixer. The affinitymatrix was spun down in a microcentrifuge for 2 min and the supernatantremoved by aspiration. Non-specifically bound ¹²⁵I-PDGF-BB was removedby resuspension of the pellet in 0.5 ml PDGF binding buffer, mixing byrotation for 5 min, centrifugation and aspiration of the supernatant,followed by two further washes with 0.5 ml 0.1% BSA, 0.2% Triton-X-100.The pellet finally obtained was resuspended in 100 ul PDGF bindingbuffer and counted in a Packard gamma counter. For displacement studies,unlabelled PDGF-BB (Amersham International) was added to the statedconcentration for the incubation of ¹²⁵I-PDGF-BB with phage.

1₂₅1-PDGF-BB bound to the fd h-PDGFB-R phage and was immunoprecipitatedin this assay. Specific binding to receptor phage was 3.5 to 4 timeshigher than the non-specific binding with vector phage fdTPs/Bs or nophage (FIG. 19). This binding of ¹²⁵I-PDGF-BB could be displaced by theinclusion of unlabelled PDGF-BB in the incubation with phage at 37° C.(FIG. 20). At 50 nM, unlabelled PDGF-BB the binding of ¹²⁵I-PDGF-BB wasreduced to the same level as the fdTPs/Bs and no phage control. FIG. 21shows the same data, but with the non-specific binding to vectordeducted.

These results indicate that a specific saturable site for ¹²⁵I-PDGF-BBis expressed an fd phage containing cloned h-PDGFB-R DNA. Thus, thephage can display the functional extracellular domain of a cell surfacereceptor.

Example 17 Construction of Phagemid Containing GeneIII Fused with theCoding Sequence for a Binding Molecule

It would be useful to improve the transfection efficiency of thephage-binding molecule system and also to have the possibility ofdisplaying different numbers and specificities of binding molecules onthe surface of the same bacteriophage. The applicants have devised amethod that achieves both aims.

The approach is derived from the phagemid system based on pUC119[Vieira, J and Messing, J. (1987) Methods Enzymol. 153:3]. In brief,gene III from fd-CAT2 (example 5) and gene III scFv fusion from fd-CAT2scFv D1.3 (example 2) were cloned downstream of the lac promoter inseparate samples of pUC119, in order that the inserted gene III and geneIII fusion could be ‘rescued’ by M13M07 helper phage [Vieira, J andMessing, J. et supra.] prepared according to Sambrootz et al. 1989supra. The majority of rescued phage would be expected to contain agenome derived from the pUC119 plasmid that contains the bindingmolecule-gene III fusion and should express varying numbers of thebinding molecule on the surface up to the normal maximum of 3-5molecules of gene III of the surface of wild type phage. The system hasbeen exemplified below using an antibody as the binding molecule.

An fdCAT2 containing the single chain Fv form of the D1.3 antilysozymeantibody was formed by digesting fdTscFvD1.3 (example 2) with PstI andXhoI, purifying the fragment containing the scFv fragment and ligatingthis into PstI and XhoI digested fdCAT2. The appropriate clone, calledfdCAT2 scFvD1.3 was selected after plating onto 2×TY tetracycline (15μg/ml) and confirmed by restriction enzyme and sequence analysis.

Gene III from fd-CAT2 (example 5) and the gene III scFv fusion fromfd-CAT2 scFvD1.3 was PCR-amplified using the primers A and B shownbelow:

Primer A: TGC GAA GCT TTG GAG CCT TTT TTT TTG GAG ATT TTC AAC G (SEQ IDNO:34)

Primer B: CAG TGA ATT CCT ATT AAG ACT CCT TAT TAC GCA GTA TGT TAG C (SEQID NO:35)-1

Primer A anneals to the 5′ end of gene III including the ribosomebinding site is located and incorporates a Hind III site. Primer Banneals to the 3′ end of gene III at the C-terminus and incorporates twoUAA stop codons and an EcoR1 site. 100 ng of fd-CAT2 and fd-CAT2 scFvD1.3 DNA was used as templates for PCR-amplification in a total reactionvolume of 5041 as described in example 7, except that 20 cycles ofamplification were performed: 94° C. 1 minute, 50° C. 1 minute, 72° C. 3minutes. This resulted in amplification of the expected 1.2 Kb fragmentfrom fd-CAT2 and a 1.8 Kb fragment from fd-CAT2 scFv D1.3.

The PCR fragments were digested with EcoRI and Hind III, gel-purifiedand ligated into Eco-R1- and Hind III-cut and dephosphorylated pUC119DNA and transformed into E. coli TG1 using standard techniques (Sambrooket al., et supra). Transformed cells were plated on SOB agar (Sambrooket al. 1989 supra) containing 100 μg/ml ampicillin and 2% glucose. Theresulting clones were called pCAT-3 (derived from fd-CAT2) and pCAT-3scFv D1.3 (deriv d from fd-CAT2 scFv D1.3).

Example 18 Rescue of Anti-Lysozyme Antibody Specificity from pCAT-3 scFvD1.3 by M13KO7

Single pCAT-3 and pCAT-3 scFv D1.3 colonies were picked into 1.5 ml 2TYcontaining 100 μg/ml ampicillin and 2% glucose, and grown 6 hrs at 30°C. 3041 of these stationary cells were added to 6 mls 2YT containing 100μg/ml ampicillin and 2% glucose in 50 ml polypropylene tubes (Falcon,Becton Dickinson Labware, 1950 Williams Drive, Oxnard, Calif. USA) andgrown for 1.5 hrs at 30° C. at 380 rpm in a New Brunswick Orbital Shaker(New Brunswick Scientific Ltd., Edison House 163 Dixons Hill road, NorthMinims, Hatfield, UK). Cells were pelleted by centrifugation at 5,000 gfor 25 minutes and the tubes drained on tissue paper. The cell pelletswere then suspended in 6 mls 2TY containing 1.25×10⁹ p.f.u. ml⁻¹ M13KO7bacteriophage added. The mixture was left on ice for 5 minutes followedby growth at 35° C. for 45 minutes at 450 rpm. A cocktail was then addedcontaining 4 μl 100 μg/ml ampicillin, 0.5 μl 0.1M IPTG and 50 μl 10mg/ml kanamycin, and the cultures grown overnight at 35° C., 450 rpm.

The following day the cultures were centrifuged and phage particles PEGprecipitated as described in example 6. Phage pellets were resuspendedin 100 μl TE (tris-EDTA see example 6) and phage titred on E. coli TG1.Aliquots of infected cells were plated on 2TY containing either 100μg/ml ampicillin to select for pUC119 phage particles, or 50 μg/mlkanamycin to select for the M13 KO7 helper phage. Plates were incubatedovernight at 37° C. and antibiotic-resistant colonies counted:

DNA amp^(R) kan^(R) pCAT-3 1.8 × 10¹¹ colonies 1.2 × 10⁹ coloniespCAT-3scFv D1.3 2.4 × 10¹¹ colonies 2.0 × 10⁹ colonies

This shows that the amp^(R) phagemid particles are infective and presentin the rescued phage population at a 100-fold excess over kan^(R) M13KO7helper phage.

Phage were assayed for anti-lysozyme activity by ELISA as described inexample 6, with the following modifications:

1) ELISA plates were blocked for 3 hrs with 2% Marvel/PBS.

2) 50 μl phage, 400 μl 1×PBS and 50 μl 20% Marvel were mixed end overend for 20 minutes at room temperature before adding 150 μl per well.

3) Phage were left to bind for 2 hours at room temperature.

4) All washes post phage binding were:

2 quick rinses PBS/0.5% Tween 20

3×2 minute washes PBS/0.5% Tween 20

2 quick rinses PBS no detergent

3×2 minute washes PBS no detergent

The result of this ELISA is shown in FIG. 22, which shows that theantibody specificity can indeed be rescued efficiently.

It is considered a truism of bacterial genetics that when mutant andwild-type proteins are co-expressed in the same cell, the wild-typeproteins are co-expressed in same cell, the wild-type protein is usedpreferentially. This is analogous to the above situation wherein mutant(i.e. antibody fusion) and wild-type gene III proteins (from M13K07) arecompeting for assembly as part of the pUC119 phagemid particle. It istherefore envisaged that the majority of the resulting pUC 119 phageparticles will have fewer gene III-antibody fusion molecules on theirsurface than is the case for purely phage system described for instancein example 2. Such phagemid antibodies are therefore likely to bindantigen with a lower avidity than fd phage antibodies with three or morecopies of the antibody fusion on their surfaces (there is no wild-typegene III, in the system described, for instance, in example 2), andprovide a route to production of phage particles with different numbersof the same binding molecule (and hence different acidities for theligand/antigen) or multiple different binding specificities on theirsurface, by using helper phage such as M13K07 to rescue cells expressingtwo or more gene III-antibody fusions.

It is also possible to derive helper phage that do not encode afunctional gene III in their genomes (by for example deleting the geneIII sequence or a portion of it or by incorporating an amber mutationwithin the gene). These defective phages will only grow on appropriatecells (for example that provide functional gene III in trans, or containan amber suppressor gene), but when used to rescue phage antibodies,will only incorporate the gene III antibody fusion encoded by thephagemid into the released phage particle.

Example 19 Transformation Efficiency of pCAT-3 and pCAT-3 scFv D1.3Phagemids

pUC19, pCAT-3 and pCAT-3 scFv D1.3 plasmid DNAs, and fdCAT-2 phage DNAwas prepared, and used to transform E. coli TG1, pCAT-3 and pCAT-3 scFvD1.3 transformations were plated on SOB agar containing 100 μg/mlampicillin and 2% glucose, and incubated overnight at 30° C. fdCAT-2transformations were plated on TY agar containing 15 μg/ml tetracyclineand incubated overnight at 37° C. Transformation efficiencies areexpressed as colonies per μg of input DNA.

DNA efficiency Transformation pUC 19 1.10⁹ pCAT-3 1.10⁸ pCAT-3scFv D1.31.10⁸ fd CAT-2 8.10⁵

As expected, transformation of the phagemid vector is approximately100-fold more efficient that the parental fdCAT-2 vector. Furthermore,the presence of a scFv antibody fragment does not compromise efficiency.This improvement in transformation efficiency is practically useful inthe generation of phage antibodies libraries that have large repertoiresof different binding specificities.

Example 20 PCR Assembly of a Single Chain Fv Library from an ImmunisedMouse

To demonstrate the utility of phage for the selection of antibodies fromrepertoires, the first requirement is to be able to prepare a diverse,representative library of the antibody repertoire of an animal anddisplay this repertoire on the surface of bacteriophage fd.

Cytoplasmic RNA was isolated according to example 14 from the pooledspleens of five male Balb/c mice boosted 8 weeks after primaryimmunisation with 2-phenyl-5-oxazolone (ph OX) coupled to chicken serumalbumin. cDNA preparation and PCR assembly of the mouse VH and VL kapparepertoires for phage display was as described in example 14. Themolecules thus obtained were ligated into fdCAT2.

Vector fdCAT2 was extensively digested with Not1 and ApaL1, purified byelectroelution (Sambrook et al. a989 supra) and 1 μg ligated to 0.5 μg(5 μg for the hierarchical libraries: see example 22) of the assembledscFv genes in 1 ml with 8000 units T4 DNA ligase (New England Biolabs).The ligation was carried out overnight at 16° C. Purified ligation mixwas electroporated in six aliquots into MC1061 cells (W. J. Dower, J. F.Miller & C. W. Ragsdale Nucleic Acids Res. 16 6127-6145 1988) and platedon NZY medium (Sambrook et al. 1989 supra) with 15 μg/ml tetracycline,in 243×243 mm dishes (Nunc): 90-95% of clones contained scFv genes byPCR screening. Recombinant colonies were screened by PCR (conditions asin example 7 using primers VH1BACK and MJK1FONX, MJK2FONX, MJK4FONX andMJK5FONX (see example 14) followed by digestion with the frequentcutting enzyme BstN1 (New England Biolabs, used according to themanufacturers instructions). The library of 2×10⁵ clones appeareddiverse as judged by the variety of digestion patterns seen in FIG. 23(i) and FIG. 23( ii), and sequencing revealed the presence of most VHgroups (R. Dildrop, Immunol. Today 5 85-86. 1984) and VK subgroups(Kabat. E. A. et al. 1987 supra) (data not shown). None of the 568clones tested bound to phOx as detected by ELISA as in example 9.

Thus the ability to select antibody provided by the use of phageantibodies (as in example 21) is essential to readily isolate antibodieswith antigen binding activity from randomly combined VH and VL domains.Very extensive screening would be required to isolate antigen-bindingfragments if the random combinatorial approach of Huse et al. 1989(supra) were used.

Example 21 Selection of Antibodies Specific for 2-Phenyl-5-Oxazolonefrom a Repertoire Derived from an Immunised Mouse

The library prepared in example 20 was used to demonstrate that abilityof the phage system to select antibodies on the basis of their antibodyspecificity.

None of the 568 clones tested from the unselected library bound to phOxas detected by

ELISA.

Screening for binding of the phage to hapten was carried

out by ELISA: 96-well plates were coated with 10 μg/ml phOx-BSA or 10μg/ml BSA in phosphate-buffered saline (PBS) overnight at roomtemperature. Colonies of phage-transduced bacteria were inoculated into200 μl 2×TY with 12.5 μg/ml tetracycline in 96-well plates (‘cellwells’, Nuclon) and grown with shaking (300 rpm) for 24 hours at 37° C.At this stage cultures were saturated and phage titres were reproducible(10¹⁰ TU/ml). 50 μl phage supernatant, mixed with 50 μl PBS containing4% skimmed milk powder, was then added to the coated plates. Furtherdetails as in example 9.

The library of phages was passed down a phOx affinity column (Table 4A),and eluted with hapten. Colonies from the library prepared in example 22were scraped into 50 ml 2×TY medium³⁷ and shaken at 37° C. for 30 min.Liberated phage were precipitated twice with polyethylene glycol andresuspended to 10¹² TU (transducing units)/ml in water (titred as inexample 8). For affinity selection, a 1 ml column of phOx-BSA-Sepharose(O. Makela, M. Kaartinen, J. L. T. Pelonen and K. Karjalainen J. Exp.Med. 148 1644-1660, 1978) was washed with 300 ml phosphate-bufferedsaline (PBS), and 20 ml PBS containing 2% skimmed milk powder (MPBS).10¹² TU phage were loaded in 10 ml MPBS, washed with 10 ml MPBS andfinally 200 ml PBS. The bound phage were eluted with 5 ml 1 mM4-ε-amino-caproic acid methylene 2-phenyl-oxazol-5-one (phOx-CAP; O.Makela et al. 1978, supra). About 10⁶ TU eluted phage were amplified byinfecting 1 ml log phase E. coli TG1 and plating as above. For a furtherround of selection, colonies were scraped into 10 ml 2×TY medium andthen processed as above. Of the eluted clones, 13% were found to bind tophOx after the first round selection, and ranged from poor to strongbinding in ELISA.

To sequence clones, template DNA was prepared from the supernatants of10 ml cultures grown for 24 hours, and sequenced using the dideoxymethod and a Sequenase kit (USB), with primer LINKFOR (see example 14)for the VH genes and primer fdSEQ1 (5′-GAA TTT TCT GTA TGA GG-3′) (SEQID NO:36) for the Vk genes. Twenty-three of these hapten-binding cloneswere sequenced and eight different VH genes (A to H) were found in avariety of pairings with seven different Vk genes (a to g) (FIG. 24).Most of the domains, such as VH-B and Vk-d were ‘promiscuous’, able tobind hapten with any of several partners.

The sequences of the V-genes were related to those seen in the secondaryresponse to phOx, but with differences (FIG. 24). Thus phOx hybridomasfrom the secondary response employ somatically mutated derivatives ofthree types of Vk genes—Vkox1. ‘Vkox-like’ and Vk45.1 genes (C. Berek,G. M. Griffiths & C. Milstein Nature 316 412-418 (1985). These can pairwith VH genes from several groups, from Vkox1 more commonly pairs withthe VHox1 gene (VH group 2. R. Dildrop supra). Vkox1 genes are always,and Vkox-like genes often, found in association with heavy chains(including VHox1) and contain a short five residue CDR3, with thesequence motif Asp-X-Gly-X-X (SEQ ID NO:37) in which the central glycineis needed to create a cavity for phOx. In the random combinatoriallibrary however, nearly all of the VH genes belonged to group 1, andmost of the Vk genes were ox-like and associated with VH domains with afive residue CDR3, motif Asp/Asn-X-Gly-X-X (SEQ ID NO:38) (FIG. 24).Vkox1 and VHox1 were found only once (Vk-f and VH-E), and not incombination with each other. Indeed Vk-f lacks the Trp91 involved inphOx binding and was paired with a VH (VH-C) with a six residue CDR3.

A matrix combination of VH and VK genes was identified in phOx-bindingclones selected from this random combinational library. The number ofclones found with each combination are shown in FIG. 25. The binding tophOx-BSA, as judged by the ELISA signal, appeared to vary (marked byshading in FIG. 25). No binding was seen to BSA alone.

A second round of selection of the original, random combinationallibrary from immune mice resulted in 93% of eluted clones binding phOx(Table 4). Most of these clones were Vk-d combinations, and boundstrongly to phOx in ELISA (data not shown). Few weak binders were seen.This suggested that affinity chromatography had not only enriched forbinders, but also for the best.

Florescence quench titrations determined the Kd of VH-B/Vk-d forphOx-GABA as 10⁻⁸ M (example 23), indicating that antibodies withaffinities representative of the secondary response can be selected fromsecondary response, only two (out of eleven characterised) secreteantibodies of a higher affinity than VH-B/Vk-d (C. Berek et al. 1985supra). The Kd of VH-B/Vk-b for phOx-GABA was determined as 10⁻⁵ M(example 23). Thus phage bearing scFv fragments with weak affinities canbe selected with antigen, probably due to the avidity of the multipleantibody heads on the phage.

This example shows that antigen specificities can be isolated fromlibraries derived from immunised mice. It will often be desired toexpress these antibodies in a soluble form for further study and for usein therapeutic and diagnostic applications. Example 23 demonstratesdetermination of the affinity of soluble scFv fragments selected usingphage antibodies. Example 27 demonstrates that soluble fragments havesimilar properties to those displayed on phage. For many purposes itwill be desired to construct and express an antibody molecule whichcontains the Fc portions of the heavy chain, and perhaps vary theimmunoglobulin isotype. To accomplish this, it is necessary to subclonethe antigen binding sites identified using the phage selection systeminto a vector for expression in mammalian cells, using methodologysimilar to that described by Orlandi, R. et al. (1989, supra). Forinstance, the VH and VL genes could be amplified separately by PCR withprimers containing appropriate restriction sites and inserted intovectors such as pSV-gpt HuIgG1 (L. Riechmann et al Nature 332 323-327),1988) which allows expression of the VH domain as part of a heavy chainIgG1 isotype and pSV-hyg HuCK which allows expression of the VL domainattached to the K light chain constant region. Furthermore, fusions ofVH and VL domains can be made with genes encoding non-immunoglobulinproteins, for example, enzymes.

Example 22 Generation of Further Antibody Specificities by the Assemblyof Hierarchical Libraries

Further antibody specificities were derived from the library preparedand screened in examples 20 and 21 using a hierarchical approach.

The promiscuity of the VH-B and Vk-d domains prompted the applicants toforce further pairings, by assembling these genes with the entirerepertoires if either Vk or VH genes from the same immunised mice. Theresulting ‘hierarchical’ libraries, (VH-B×Vk-rep and VH-rep×Vk-d), eachwith 4×10⁷ members, were subjected to a round of selection andhapten-binding clones isolated (Table 4). As shown by ELISA, most werestrong binders. By sequencing twenty-four clones from each library, theapplicants identified fourteen new partners for VH-B and thirteen forVk-d (FIG. 24). Apart from VH-B and Vk-c, none of the previous partners(or indeed other clones) from the random combinatorial library wasisolated again. Again the Vk genes were mainly α-like and the VH genesmainly group 1 (as defined in Dildrop, R. 1984 supra), but the onlyexamples of Vkox1 (Vk-h, -p, -q and -r) have Trp91, and the VH-CDR3motif Asp-X-Gly-X-X (SEQ ID NO:37) now predominates. Thus some featuresof the phOx hybridomas seemed to emerge more strongly in thehierarchical library. The new partners differed from each other mainlyby small alterations in the CDRs, indicating that much of the subtlediversity had remained untapped by the random combinatorial approach.More generally it has been shown that a spectrum of related antibodiescan be made by keeping one of the partners fixed and varying the other,and this could prove invaluable for fine tuning of antibody affinity andspecificity.

Therefore, again, phage antibodies allow a greater range of antibodymolecules to be analysed for desired properties.

This example, and example 21, demonstrate the isolation of individualantibody specificities through display on the surface of phage. However,for some purposes it may be more desirable to have a mixture ofantibodies, equivalent to a polyclonal antiserum (for instance, forimmunoprecipitation). To prepare a mixture of antibodies, one could mixclones and express soluble antibodies or antibody fragments oralternatively select clones from a library to give a highly enrichedpool of genes encoding antibodies or antibody fragments directed againsta ligand of interest and express antibodies from these clones.

Example 23 Selection of Antibodies Displayed on Bacteriophage withDifferent Affinities for 2-Phenyl-5-Oxazolone using AffinityChromatography

The ELISA data shown in example 21 suggested that affinitychromatography had not only enriched for binders, but also for the best.To confirm this, the binding affinities of a strong binding and a weakbinding phage were determined and then demonstrated that they could beseparated from each other using affinity chromatography.

Clones VH-B/Vk-b and VH-B/Vk-d were reamplified with MJK1FONX, MJK2FONX,MJK4FONX and MJK5FONX (see example 14) and VH1BACK-SfiI (5′-TCG CGG CCCAGC CGG CCA TGG CC(G/C) AGG T(C/G)(A/C) A(A/G)C TGC AG(C/G) AGT C(A/T)GG-3′) (SEQ ID NO:39), a primer that introduces an SfiI site (underlined)at the 5′ end of the VH gene. VH-B/Vk-d was cloned into a phagemid e.g.pJM1 (a gift from A. Griffiths and J. Marks) as an SfiI-NotI cassette,downstream of the pelB leader for periplasmic secretion (M. Better atal. supra), with a C-terminal peptide tag for detection (see example 24and figure), and under the control of a P_(L) promoter (H. Shimatake &M. Rosenberg Nature 292 128-132 1981). The phagemid should have thefollowing features: a) unique SfiI and NotI restriction sites downstreamof a pelB leader; b) a sequence encoding a C-terminal peptide tag fordetection; and c) a λP_(L) promoter controlling expression. 10 litercultures of E. coli N4830-1 (M. E. Gottesman, S. Adhya & A. Das J. Mol.Biol. 140 57-75 1980) harbouring each phagemid were induced as in K.Nagai & H. C. Thogerson (Methods Enzymol 153 461-481 1987) andsupernatants precipitated with 50% ammonium sulphate. The resuspendedprecipitate was dialysed into PBS+0.2 mM EDTA (PBSE), loaded onto a 1.5ml column of phOx:Sepharose and the column washed sequentially with 100ml PBS: 100 ml 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0: 10 ml 50 mM citrate,pH 5.0: 10 ml 50 mM citrate, pH4.0, and 20 ml 50 mM glycine, pH 3.0.scFv fragments were eluted with 50 mM glycine, pH 2.0, neutralised withTris base and dialysed against PBSE. VH-B/Vk-b was cloned into aphagemid vector based on pUC119 encoding identical signal and tagsequences to pJM1, and expression induced at 30° C. in a 10 literculture of E. coli TG1 harbouring the phagemid as in D. de Bellis & I.Schwartz (1980 Nucleic Acids Res 18 1311). The low affinity of cloneVH-B/Vk-b made its purification on phOx-Sepharose impossible. Thereforeafter concentration by ultrafiltration (Filtron, Flowgen), thesupernatant (100 ml of 600 ml) was loaded onto a 1 ml column of proteinA-Sepharose coupled (E. Harlow & D. Lane 1988 supra) to the monoclonalantibody 9E10 (Evan, G. I. et al. Mol. Cell Biol. 5 3610-3616 1985) thatrecognises the peptide tag. The column was washed with 200 ml PBS and 50ml PBS made 0.5 M in NaCl. scFv fragments were eluted with 100 ml 0.2Mglycine, pH 3.0, with neutralisation and dialysis as before.

The Kd (1.0±0.2×10⁸ M) for clone VH-B/Vk-d was determined byfluorescence quench titration with 4-E-amino-butyric acid methylene2-phenyl-oxazol-5-one (phOx-GABA Co. Makela et al, 1978 supra).Excitation was at 280 nm, emission was monitored at 340 nm and the K_(d)calculated. The K_(d) of the low affinity clone VH-B/Vk-b was determinedas 1.8±0.3×10⁻⁵ M (not shown). To minimise light adsorption by thehigher concentrations of phOx-GABA required, excitation was at 260 nmand emission was monitored at 304 nm. In addition the fluorescencevalues were divided by those from a parallel titration of the lysozymebinding Fv fragment D1.3. The value was calculated as in H. N. EisenMeth. Med. Res. 10 115-121 1964. A mixture of clones VH-B/Vk-b andVH-B/Vk-d, 7×10¹⁰ TU phage in the ratio 20 VH-B/Vk-b: 1 VH-B/Vk-d wereloaded onto a phOx-BSA-Sepharose column in 10 ml MPBS and eluted asabove. Eluted phage were used to reinfect E. coli TG1, and phageproduced and harvested as before. Approximately 10¹¹ TU phage wereloaded onto a second affinity column and the process repeated to give atotal of three column passes. Dilutions of eluted phage at each stagewere plated in duplicate and probed separately with oligonucleotidesspecific for Vk-b (5′-GAG CGG GTA ACC ACT GTA CT-3′) (SEQ ID NO:40) orVk-d (5′-GAA TGG TAT AGT ACT ACC CT-3′) (SEQ ID NO:41). After these tworounds, essentially all the eluted phage were VH-B/Vk-d (table 4).Therefore phage antibodies can be selected on the basis of the antigenaffinity of the antibody displayed.

Example 24 Construction of Phagemid pHEN1 for the Expression of AntibodyFragments Expressed on the Surface of Bacteriophage followingSuperinfection

The phagemid pHEN1 (FIG. 26( a)) is a derivative of pUC119 (Vieira, J. &Messing, J. Methods Enzymol 153 pp 3-11, 1987). The coding region of g3pfrom fdCAT2, including signal peptide and cloning sites, was amplifiedby PCR, using primers G3FUFO and G3FUBA (given below) (which containEcoRI and HindIII sites respectively), and cloned as a HindIII-EcoRIfragment into pUC119. The HindIII-NotI fragment encoding the g3p signalsequence was the replaced by a pelB signal peptide (Better, M. et al.Science 240 1041-1043, 1988) with an internal SfiI site, allowingantibody genes to be cloned as fiI-NotI fragments. A peptide tag, c-myc,(Munro, S. & Pelham, H. Cell 46 291-300, 1986) was introduced directlyafter the NotI site by cloning an oligonucleotide cassette, and followedby an amber codon introduced by site-directed mutagenesis using an invitro mutagenesis kit (Amersham International) (FIG. 26( b)).

G3FUFO, 5′-CAG TGA ATT CTT ATT AAG ACT CCT TAT TAC GCA GTA TGT TAG C-3′(SEQ ID NO:42);

G3FUBA, 5′-TGC GAA GCT TTG GAG CCT TTT TTT TTG GAG ATT TTC AAC G-3′ (SEQID NO:43);

Example 25 Display of Single Chain Fv and Fab Fragments Derived from theAnti-Oxazolone Antibody NQ10.12.5 on Bacteriophage fd using pHEN1 andfdCAT2

A range of constructs (see FIG. 27) were made from a clone (essentiallyconstruct II in pUC19) designed for expression in bacteria of a solubleFab fragment (Better et al. 1988 see above) from the mouse anti-phOx(2-phenyl-5-oxazolone) antibody NQ10.12.5 (Griffiths, G. M. et al.Nature 312, 271-275, 1984). In construct II, the V-regions are derivedfrom NQ10.12.5 and attached to human Ck and CH1 (γ1 isotype) constantdomains. The C-terminal cysteine residues, which normally form acovalent link between light and heavy antibody chains, have been deletedfrom both the constant domains. To clone heavy and light chain genestogether as Fab fragments (construct II) or as separate chains(constructs III and IV) for phage display, DNA was amplified fromconstruct II by PCR to introduce a NotI restriction site at the 3′ end,and at the 5′ end either an ApaLI site (for cloning into fd-CAT2) orSfiI site (for cloning into pHEN1). The primers FABNOTFOK withVH1BACKAPA (or VH1BACKSFI15) were used for PCR amplification of genesencoding Fab fragments (construct II), the primers FABNOTFOH withVH1BACKAPA (or VH1BACKSFI15) for heavy chains (construct III), and theprimers FABNOTFOK and MVKBAAPA (or MVKBASFI) for light chains (constructIV).

The single-chain Fv version of NQ10.12.5 (construct I) has the heavy(VH) and light chain (Vk) variable domains joined by a flexible linker(Gly₄Ser)₃ (Huston, J. S. et al. Proc. Natl. Acad. Sci. USA 855879-5883, 1988) and was constructed from construct II by ‘splicing byoverlap extension’ as in example 14. The assembled genes werereamplified with primers VK3F2NOT and VH1BACKAPA (or VH1BACKSFI15) toappend restriction sites for cloning into fd-CAT2 (ApaLI-NotI) or pHEN1(SfiI-NotI).

VH1BACKAPA, 5′-CAT GAC CAC AGT GCA CAG GT(C/G) (A/C)A(A/G) CTG CAG(C/G)AG TC(A/T) GG-3′ (SEQ ID NO:44);

VH1BACKSFI15, 5′-CAT GCC ATG ACT CGC GGC CCA GCC GGC CAT GGC C(C/G)A GGT(C/G)(A/C)A (A/G)CT GCA G(C/G)A GTC (A/T)GG-3′ (SEQ ID NO:45);

FABNOTFOH, 5′-CCA CGA TTC TGC GGC CGC TGA AGA TTT GGG CTC AAC TTT CTTGTC GAC-3′ (SEQ ID NO:46);

FABNOTFOK, 5′-CCA CGA TTC TGC GGC CGC TGA CTC TCC GCG GTT GAA GCT CTTTGT GAC-3′ (SEQ ID NO:47);

MVKBAAPA, 5′-CAC AGT GCA CTC GAC ATT GAG CTC ACC CAG TCT CCA-3′ (SEQ IDNO:48);

MVKBASFI, 5′-CAT GAC CAC GCG GCC CAG CCG GCC ATG GCC GAC ATT GAG CTC ACCCAG TCT CCA-3′ (SEQ ID NO:49);

VK3F2NOT, 5′-TTC TGC GGC CGC CCG TTT CAG CTC GAG CTT GGT CCC-3′ (SEQ IDNO:50).

Restriction sites are underlined.

Rescue of Phage and Phagemid Particles

Constructs I-IV (FIG. 27) were introduced into both fd-CAT2 and pHEN1.Phage fd-CAT2 (and fd-CAT2-I, II, III or IV) was taken from thesupernatant of infected E. coli TG1 after shaking at 37° C. overnight in2×TY medium with 12.5 μg/ml tetracycline, and used directly in ELISA.Phagemid pHEN1 (and pHEN1-I and II) in E. coli TG1 (supE) were grownovernight in 2 ml 2×TY medium, 100 μg/ml ampicillin, and 1% glucose(without glucose, expression of g3p prevents later superinfection byhelper phage). 10 μl of the overnight culture was used to innoculate 2ml of 2×TY medium, 100 μg/ml ampicillin, 1% glucose, and shaken at 37°C. for 1 hour. The cells were washed and resuspended in 2×TY, 100 μg/mlampicillin, and aphagemid particles rescued by adding 2 μl (10⁸ pfu)VCSM13 helper phage (Stratagene). After growth for one hour, 4 μlkanamycin (25 mg/ml) was added, and the culture grown overnight. Thephagemid particles were concentrated 10-fold for ELISA by precipitationwith polyethylene glycol.ELISA

Detection of phage binding to 2-phenyl-5-oxazolone (phOx) was performedas in example 9. 96-well plates were coated with 10 μg/ml phOx-BSA or 10μg/ml BSA in PBS overnight at room temperature, and blocked with PBSScontaining 2% skimmed milk powder. Phage (mid) supernatant (50 μl) mixedwith 50 μl PBS containing 4% skimmed milk powder was added to the wellsand assayed. To detect binding of soluble scFv or Fab fragments secretedfrom pHEN1, the c-myc peptide tag described by Munro and Pelham 1986supra, was detected using the anti-myc monoclonal 9E10 (Evan, G. I. etal. Mol Cell Biol 5 3610-3616, 1985) followed by detection withperoxidase-conjugated goat anti-mouse immunoglobulin. Other details areas in example 9.

The constructs in fdCAT2 and pHEN1 display antibody fragments of thesurface of filamentous phage. The phage vector, fd-CAT2 (FIG. 8) isbased on the vector fd-tet (Zacher, A. N. et al. Gene 9 127-140, 1980)and has restriction sites (ApaLI and NotI) for cloning antibody genes(or other protein) genes for expression as fusions to the N-terminus ofthe phage coat protein g3p. Transcription of the antibody-g3p fusions infd-CAT2 is driven from the gene III promoter and the fusion proteintargeted to the periplasm by means of the g3p leader. Fab abd scFvfragments of NQ10.12.5 cloned into fd-CAT2 for display were shown tobind to phOx-BSA (but not BSA) by ELISA (table 5). Phage were consideredto be binding if A₄₀₅ of the sample was at least 10-fold greater thatthe background in ELISA.

The phagemid vector, pHEN1 (FIG. 26( a)), is based upon pUC119 andcontains restriction sites (SfiI and NotI) for cloning the fusionproteins. Here the transcription of antibody-g3p fusions is driven fromthe inducible lacZ promoter and the fusion protein targeted to theperiplasm by means of the pelB leader. Phagemid was rescued with VCSM13helper phage in 2×TY medium containing no glucose or IPTG: under theseconditions there is sufficient expression of antibody-g3p. Fab and scFvfragments of NQ10.12.5 cloned into pHEN1 for display were shown to bindto phOx-BSA (but not BSA) by ELISA (Table 5) using the same criterion asabove.

An alternative methodology for preparing libraries of Fab fragmentsexpressed on the surface of phage would be to:

1. Prepare a library of phage expressing heavy chain (VHCH) genes frominserts in the phage genome.

2. Prepare a library of light chain genes in a plasmid expression vectorin E. coli, preferably a phagemid, and isolate the soluble protein lightchains expressed from this library.

3. Bind the soluble protein light chains from the library to the heavychain library displayed on phage.

4. Select phage with the desired properties of affinity and specificity.

These will encode the heavy chain (VHCH) genes.

5. Isolate the light chain genes encoding light chains which formsuitable antigen binding sites in combination with the selected heavychains, preferably by using superinfectin of bacteria, containingphagemid expressing the light chain, with phage expressing the selectedheavy chain (as described in example 20) and then assaying for antigenbinding.

Example 26 Rescue of Phagemid Encoding a Gene III Protein Fusion withAntibody Heavy or Light Chains by Phage Encoding the ComplementaryAntibody Chain Displayed on Phage and the Use of this Technique to MakeDual Combinatorial Libraries

With random combinatorial libraries there is a limitation on thepotential diversity of displayed Fab fragments due to the transformationefficiency of bacterial cells. Described here is a strategy (dualcombinatorial libraries) to overcome this problem, potentiallyincreasing the number of phage surveyed by a factor of 10⁷.

For assembly of heavy and light chains expresses from different vectors,phagemid (pHEN1-III or IV) was grown in E. coli HB2151 (a non-suppressorstrain) to allow production of soluble chains, and rescued as above(example 27) except that helper phage were used expressing partnerchains as fusions to g3p (10⁹ TU fd-CAT2-IV or III respectively) and 2μl tetracycline (12.5 mg/ml) in place of kanamycin.

Separate Vectors to Encode Fab Heavy and Light Chains

The heavy and light chains of Fab fragments can be encoded together inthe same vector (example 25) or in different vectors. To demonstratethis the heavy chain (construct III) was cloned into pHEN1 (to providesoluble fragments) and the light chain (construct IV) into fd-CAT2 (tomake the fusion with g3p). The phagemid pHEN1-III, grown in E. coliHB2151 (non-suppressor) was rescued with fd-CAT2-IV phage, andphage(mid) shown to bind to phOx:BSA, but not to BSA (Table 5). Thisdemonstrates that soluble light chain is correctly associating with theheavy chain anchored to the g3p, since neither heavy chain nor lightchain alone bind antigen (Table 5).

Similar results were obtained in the reverse experiment (with phagemidpHEN-1-IV and fd-CAT2-III phage) in which the heavy chain was producedas a soluble molecule and the light chain anchored to g3p (Table 5).Hence a Fab fragment is assembled on the surface of phage by fusion ofeither heavy or light chain to g3p, provided the other chain is secretedusing the same or another vector (FIG. 28).

The resulting phage population is a mixture of phage abd rescuedphagemid. The ratio of the two types of particle was assessed byinfecting log phase E. coli TG1 and plating on TYE plates with either 15μg/ml tetracycline (to select for fd-CAT2) or 100 μg/ml ampicillin (toselect for pHEN1). The titre of fd-CAT2 phage was 5×10¹¹ TU/ml and thetitre of pHEN1 2×10¹⁰ TU/ml, indicating a packaging ratio of 25 phageper phagemid.

Demonstrated here is an alternative strategy involving display of theheterodimeric antibody Fab fragments on the surface of phage. One of thechains is fused to g3p and the other is secreted in soluble form intothe periplasmic space of the E. coli where it associates non-covalentlywith the g3p fusion, and binds specifically to antigen. Either the lightor heavy chain can be fused to the g3p: they are displayed on the phageas Fab fragments and bind antigen (FIG. 28). Described are both phageand phagemid vectors for surface display. Phagemids are probablysuperior to phage vectors for creation of large phage display libraries.Particularly in view of their higher transfection efficiencies (Two tothree orders of magnitude higher), allowing larger libraries to beconstructed. The phagemid vector, pHEN1 also allows the expression ofsoluble Fab fragments in non-suppressor E. coli.

Also demonstrated here is that heavy and light chains encoded on thesame vector (construct II), or on different vectors (constructs III andIV) can be displayed as Fab fragments. This offers two distinct ways ofmaking random combinatorial libraries for display. Libraries of heavyand light chain genes, amplified by PCR, could be randomly linked by a‘PCR assembly’ process (example 14) based on ‘splicing by overlapextension’, cloned into phage(mid) display vectors and expressed fromthe same promoter as part of the same transcript (construct II) asabove, or indeed from different promoters as separate transcripts. Herethe phage(mid) vector encodes and displays both chains. For acombinatorial library of 10⁷ heavy chains and 10⁷ light chains, thepotential diversity of displayed Fab fragments (10¹⁴) is limited by thetransfection efficiency of bacterial cells by the vector (about 10⁹clones per μg cut and ligated plasmid at best) (W. J. Dower et al Nucl.Acids. Res. 16 6127-6145, 1988). Libraries thus prepared are analogousto the random combinatorial library method described by Huse, W. D. etal Science 246 1275-1281 (1989), but have the important additionalfeature that display on the surface of phage gives a powerful method ofselecting antibody specificities from the large number of clonesgenerated.

Alternatively, libraries of heavy and light chains could be cloned intodifferent vectors for expression in the same cell, with a phage vectorencoding the g3p fusion and a phagemid encoding the soluble chain. Thephage acts as a helper, and the infected bacteria produced both packagedphage and phagemid. Each phage or phagemid displays both chains butencodes only one chain and thus only the genetic information for half ofthe antigen-binding site. However, the genes for both antibody chainscan be recovered separately by plating on the selective medium,suggesting a means by which mutually complementary pairs of antigenbinding heavy and light chain combinations could be selected from randomcombinatorial libraries. For example, a light chain repertoire on fdphage could be used to infect cells harbouring a library of solubleheavy chains on the phagemid. The affinity purified phagemid librarycould then be used to infect E. coli, rescued with the affinity purifiedphage library, and the new combinatorial library subjected to a furtherround of selection. Thus, antibody heavy and light chain genes arereshuffled after each round of purification. Finally, after severalrounds, infected bacteria could be plated and screened individually forantigen-binding phage. Such ‘dual’ combinatorial libraries arepotentially more diverse than those encoded on a single vector. Bycombining separate libraries of 10⁷ light chain phage(mid)s, thediversity of displayed Fab fragments (potentially 10¹⁴) is limited onlyby the number of bacteria (10¹² per liter). More simply, the use of twovectors should also facilitate the construction of ‘hierarchical’libraries, in which a fixed heavy or light chain is paired with alibrary or partners (example 22), offering a means of ‘fine-tuning’antibody affinity and specificity.

Example 27 Induction of Soluble scFv and Fab Fragments Using PhagemidpHEN1

Further study of antibodies which have been expressed on the surface ofphage would be greatly facilitated if it is simple to switch toexpression in solution.

E. coli HB2151 was infected with pHEN phagemid (pHEN1-I or II), andplated on YTE, 100 μg/ml ampicillin plates. Colonies were shaken at 37°C. in 2×TY medium, 100 μg/ml ampicillin, 1% glucose to OD₅₅₀=0.5 to 1.0.Cells were pelleted, washed once in 2×TY medium, resuspended in mediumwith 100 μg/ml ampicillin, 1 mM isopropyl β-D-thiogalactoside (IPTG),and grown for a further 16 hours. Cells were pelleted and thesupernatant, containing the secreted chains, used directly in ELISA.

The phagemid pHEN1 has the advantage over phage fd-CAT2, in thatantibody can be produced either for phage display (by growth in supEstrains of E. coli) or as a tagged soluble fragment (by growth innon-suppressor strains), as a peptide tag (example 24) and amber codonwere introduced between the antibody and g3p. Secretion of soluble Fabfragments from pHEN1-II or scFv fragments from pHEN1-I was demonstratedafter growth in E. coli HB2151 and induction with IPTG using Westernblots (FIG. 29). For detection of secreted proteins, 10 μl supernatantof induced cultures were subjected to SDS-PAGE and proteins transferredby electroblotting to Immobilon-P (Millipore). Soluble heavy and lightchain were detected with goat polyclonal anti-human Fab antiserum(Sigma) and peroxidase conjugated rabbit anti-goat immunoglobulin(Sigma), each at a dilution of 1:1000. The tagged VK domain was detectedwith 9E10 antibody (1:1000) and peroxidase conjugated goat anti-mouseimmunoglobulin (Fc specific) (1:1000) (Sigma) or with a peroxidaselabelled anti-human CK antiserum (Dako). 3,3′-diaminobenzidine (DAB;Sigma) was used as peroxidase substrate (Harlow E., et al. 1988 Supra).With the scFv, the fragments were detected using the 9E10 anti-myc tagantibody (data not shown). With the Fab, only the light chain wasdetected by 9E10 (or anti-human CK) antibody, as expected, while theanti-human Fab antiserum detected both heavy and light chains. Bindingof the soluble scFv and Fab fragments to phOx-BSA (but not to BSA) wasalso demonstrated by ELISA (Table 58). Thus scFv and Fab fragments canbe displayed on phage or secreted as soluble fragments from the samephagemid vector.

Example 28 Increased Sensitivity in ELISA assay of Lysozyme UsingFDTscFvD1.3 as Primary Antibody Compared to Soluble scFvD1.3

In principle the use of phage antibodies should allow more sensitiveimmunoassays to be performed than with soluble antibodies. Phageantibodies combine the ability to bind a specific antigen with thepotential for amplification through the presence of multiple (ca.2800)copies of the major coat protein (g8p) on each virion. This would allowthe attachment of several antibody molecules directed against M13 toeach virion followed by the attachment of several molecules ofperoxidase-conjugated anti-species antibody (anti-sheep) IgG in the casebelow). Thus for every phage antibody bound to antigen there is thepotential for attaching several peroxidase molecules whereas when asoluble antibody is used as the primary antibody this amplification willnot occur.

ELISA plates were coated overnight at room temperature using 200 μl of10 fold dilutions of hen egg lysozyme (1000, 100, 10, 1, 0.1 and 0.01μg/ml) in 50 mM NaHCO₃, pH9.6. ELISA was performed as described inexample 4 except that (i) incubation with anti-lysozyme antibody waswith either

FDTscFvD1.3 (pAb; 10¹¹ phage per well; 1.6 mol) or soluble affinitypurified scFvD1.3 (18 μg per well; 0.7 nmol) (ii) incubation with secondantibody was with 1/100 dilution of sheep anti-M13 serum for FDTscFvD1.3samples or with or 1/100 dilution of rabbit anti-scFvD1.3 serum (from S.Ward) for soluble scFvD1.3 samples (iii) peroxidase-conjugated rabbitanti-goat immunoglobulin (Sigma; 1/5000) was used for FDTscFvD1.3samples and peroxidase-conjugated goat anti-rabbit immunoglobulin(Sigma; 1/5000) was used for soluble scFvD1.3 samples. Absorbance at 405nm was measured after 15 h. The results are shown in FIGS. 30 and 31. Inthese figures lysozyme concentrations for coating are shown on a logscale of dilutions relative to 1 μg/ml. (i.e. log=−3=1 mg/ml; log=2=0.01μg/ml)

Higher signals were obtained with FDTscFvD1.3 at all concentrations oflysozyme (FIG. 31) but the difference was very marked at the greatestdilutions, where antigen quantities are most limiting (FIGS. 30 and 31).This suggests that phage antibodies may be particularly valuable forsandwich type assays where the capture of small amounts of antigen bythe primary antibody will generate an amplified signal when phageantibodies directed against a different epitope are used as the secondantigen binding antibody.

Example 29 Direct Rescue and Expression of Mouse Monoclonal Antibodiesas Single Chain Fv Fragments on the Surface of Bacteriophage fd

The principle is very similar to that described in example 14. Itconsists of the PCR assembly of single chain antibodies from cDNAprepared from mouse monoclonals. As an example, the rescue andexpression of two such antibodies from monoclonals expressing antibodiesagainst the steroid hormone oestriol is described.

A. RNA Preparation

RNA can be prepared using many procedures well known to those skilled inthe art. In this example, the use of Triton X-100 lysis, phenol/SDSRNase inactivation gave excellent results.

1. The mouse monoclonal cells that were used here had been harvested bycentrifugation and resuspended in serum free medium. They were thencentrifuged and resuspended in saline and after a final centrifugationstep, resuspended in sterile water at 1×10⁷ cells per ml. (Normallycells would be washed in PBS buffer and finally resuspended in PBSbuffer, but these particular cells were supplied to us as describedfrozen in water).2. To 750 μl of cells was added 250 ul of ice cold 4× lysis buffer (40mM Tris HCl pH 7.4/4 mM MgCl₂/600 mM NaCl/40 mM VRC (Veronyl ribosylcomplex)/2% Triton X-100). The suspension was mixed well and left on icefor 5 minutes.3. Centrifugation was carried out at 4° C. in a microfuge at 13000 rpmfor 5 min.The supernatant is then phenol extracted three times, phenol chloroformextracted three times and finally, ethanol precipitated as described inthe materials and methods. The precipitate was resuspended in 50 ulwater.4. The optical density of the RNA at 260 nm with a 2.5 ul sample in 1 mlwater was measured. The RNA was checked by electrophoresis of a 2 ugsample on a 1% agarose gel. RNA in the range of 32 ug to 42 ug wasobtained by this method.B. cDNA Preparation

The method used is the same as that described in example 14. Two cDNApreparations were made. These were from RNA extracted from themonoclonals known as cell lines 013 and 014 which both expressantibodies against the steroid hormone, oestriol.

C. Primary PCRs

The method used is essentially the same as that described in example 14.The VH region was amplified with the primers VH1BACK and VH1FOR-2. Forthe Vkappa region, four separate reactions were carried out using theprimer VK2BACK and wither MJK1FONX, MJK2FONX, MJK4FONX or MJK5FONX.Samples (5 ul) were checked on a 1.5% agarose gel. From this it wasobserved that for cDNA prepared from the two oestriol monoclonals theprimers VK2BACK and MJK1FONX gave the best amplification of the Vkapparegion. The VH bands and the Vkappa bands amplified withVK2BACK/MJK1FONX were purified on 2% low melting point agarose gels foreach monoclonals. The DNA bands were excised from the gel and purifiedusing a dedicated Geneclean kit as described in example 14.

D. Preparation of Linker

The method used is essentially the same as that described in example 14.In this case, the amplified linker DNA was purified on a 2% agarose geland recovered from the gel with a dedicated “Mermaid” kit (BIO 101,Geneclean, La Jolla, San Diego, Calif., USA) using the manufacturersinstructions.

E. Assembly PCRs

The method used is essentially the same as that described in example 14.In this case, the assembled

PCR product was purified on a 2% agarose gel and recovered from the gelwith a dedicated “Mermaid” kit.

F. Adding Restriction Sites and Work-Up

The assembled product was “tagged” with Apa LI and Not I restrictionsites. The DNA was then digested with Apa LI and Not I to give theappropriate sticky ends for cloning and then purified on a 2% lowmelting point agarose gel and extracted using a Geneclean kit. Themethod used is the same as that described in example 14.

G. Cloning into Vector fd-CAT2

A total of 15 ug of CsCl purified fd-CAT2 DNA was digested with 100units of the restriction enzyme Not I (New England Biolabs) in a totalvolume of 200 ul 1×NEB Not I buffer with 1×NEB acetylated BSA for atotal of 3 hours at 37° C. The vector DNA was the treated twice with 15ul STRATACLEAN (a commercially available resin for the removal ofprotein), following the manufacturers instructions (Stratagene, 11099North Torrey Pines Road, La Jolla, Calif., USA). The DNA was thenethanol precipitated and redissolved in TE buffer (Sambrook et al., 1989supra). The DNA was then digested with 100 units of the restrictionenzyme Apa LI (New England Biolabs) in a total volume of 200 ul 1×NEBBuffer 4 overnight at 37° C. The vector was then purified with a ChromaSpin 1000 column following the manufacturers instructions (ClontechLaboratories Inc, 4030 Fabian way, Palo Alto, Calif., USA). This stepremoves the Apa LI/Not I fragment to give cut vector DNA for maximumligation efficiency.

Ligation reactions were carried out with 2.5-10 ng of the DNA insert andlong of vector in a total volume of 10 ul of 1×NEB ligase buffer with 1ul of NEB ligase (New England Biolabs) at 16° C. overnight (approx 16hours).

H. Transformation and Growth

E. coli strain TG1 was made competent and transformed with the fdCAT2recombinant DNA as described by Sambrook et al, 1989 Supra. The cellswere plated out on LBtet plates (10 g tryptone, 5 g yeast extract, 10 gNaCl, 15 g bacto-agar per liter with 15 ug/ul of tetracycline added justbefore pouring the plates) and grown overnight.

Single well isolated colonies were then inoculated into 10 ml of LBtetbroth (LB medium with 15 ug/ul of tetracycline) in 50 ml tubes. Afterovernight growth at 35° C./350 rpm in a bench top centrifuge. Thesupernatants were transferred to 15 ml centrifuge tubes and 2 ml 20% PEG8000/2.5M NaCl added to each. After incubating at room temperature for20-30 minutes, the recombinant phage was pelleted by centrifugation at9000 rpm in a Sorval SM24 rotor for 30 minutes. The PEG supernatant wasdiscarded. Any remaining PEG was removed with a pasteur pipette after abrief (2 minutes) centrifugation step. This last step was repeated tomake sure that no PEG remained. The phage pellet was then resuspended in500 ul PBS buffer. This was transferred to a microcentrifuge tube andspun at 13000 rpm to remove any remaining cells. The phage supernatantwas transferred to a fresh tube.

I. Assay for Antibody Expression

Bacteriophage fd recombinants were screened for the expression ofantibody against oestriol by ELISA.

This method is described in example 6. In this case the followingalterations are relevant.

1. Microtitre plates were coated overnight with 40 ug/ml oestriol-6carboxymethyloxime-BSA (Steraloids, 31 Radcliffe Road, Croydon, CRO 5QJ,England).

2. 1st antibody was the putative phage anti oestriol antibody. 50 ul ofphage in a final volume of 200 ul of sterile PBS combining 0.25% gelatinwas added to each well.

3. 2nd antibody was sheep anti M13 at 1:1000 dilution.

4. 3rd antibody was peroxidase conjugated rabbit anti goatimmunoglobulin.

Recombinants expressing functional antibody were detected by incubationwith the chromogenic substrate 2′2′ axinobis (3-ethyl benzothiazolinesulphonic acid). The results are shown in FIGS. 32 and 33.

Example 30 Kinetic Properties of Alkaline Phosphatase Displayed on theSurface of Bacteriophage fd

This example demonstrates that kinetic properties of an enzyme expressedon phage are qualitatively similar to those in solution. Bacteriophagefd displaying alkaline phosphatase fusions of gene 3 with either thenative arginine (see example 31) or the mutant residue alanine atposition 166 (see example 11) were prepared by PEG precipitation asdescribed in the materials and methods.

The kinetic parameters of alkaline phosphatase expressed on the surfaceof fd phage were investigated in 1M Tris/HCl, pH8.0 at 20° C. with 1 ml4-nitrophenyl phosphate as substrate. The reactions were initiated bythe addition of 100 μl of a phage-alkaline phosphatase fusionpreparation, 50 fold concentrated with respect to the original culturesupernatant. The rate of change of absorbance was monitored at 410 nmusing a Philips 8730 spectrophotometer and the initial reaction ratecalculated using a molar absorbance of 16200 l/mol/cm. For the fdphoAla166 enzyme but not fdphoArg166a lag phage was seen following thisaddition, the reaction rate accelerating until a steady state wasobtained after approximately 60 to 90 secs. This steady state rate wasused for determination of kinetic parameters. No deviation formMichaelis Menten kinetics was apparent for either phage enzyme. Valuesof K_(m) and k_(cat) were derived from plots of s/v against s and areshown in Table 6.

Because of the difficulty in establishing the relationship between thenumber of phage particles an the number of active enzyme dimers formedon the phage k_(cat) values are expressed not as absolute values, but asrelative values between the two enzyme forms. Western blots (carried outas in example 31 using antig3p antiserum) of the phage enzymepreparations used in this experiment showed approximately equalintensities for the full length fusion band with the Arg166 and Ala166enzymes when detected using antibody directed against gene3. In thesepreparations the intact fusion represents approximately 30% of thedetected material. The two preparations were therefore assumed to beexpressing approximately the same concentrations of intact fusions.

Table 6 summarises the kinetic data from this experiment and compares itwith data from Chaidaroglou, A. et al (Biochemistry 27, 8338-8343(1988)) obtained with soluble preparations of the wild type and mutantenzyme forms. The same substrate and assay conditions were used in bothexperiments. Soluble alkaline phosphatase was also tested in parallel inour experiments (K_(m)=8.5 μM; kcat=3480 mol substrate converted molenzyme⁻¹ min⁻¹).

The effect of mutating arginine at position 166 to alanine isqualitatively similar for the phage enzyme as for the soluble enzyme.K_(m) is increased about 15 fold and the relative k_(cat) is decreasedto 36% of that for wild type. This increased K_(m) would reflect areduction in substrate affinity in the phage enzyme on mutation ofArg166, as was proposed for the soluble enzyme (Chaidaroglou et al, 1988supra), assuming the same kinetic mechanism applies. There are, however,some quantitative differences in the behaviour of K_(m) of the phageenzyme. The K_(m) of 73 μM observed for fdphoArg166 compares with aK_(m) of 12.7 μM for the free enzyme; the K_(m) for fdphoAla166 is 1070μM whereas the free mutant enzyme has a K_(m) of 1620 μM. One canspeculate that the higher K_(m) for fdphoArg 166 and the lower K_(w) forfdphoAla166, compared to the soluble enzymes result from the ‘anchored’alkaline phosphatase fusion molecules interacting to form dimers in adifferent manner to the enzyme in free solution.

The relative values of k_(cat) for the Arg166 and Ala166 forms arehowever very similar for both the phage enzymes and the soluble enzymes,a reduction occurring on mutation to 35 to 40% of the value for thenative enzyme. The rate limiting step, determining k_(cat), for solublephoArg166 is thought to be dissociation of non-covalently boundphosphate from the enzyme (Hull W. E. et al. Biochemistry 15, 1547-15611976). Chaidaroglou et al (1988) supra suggest that, for the solubleenzyme, mutation of Arg166 to alanine alters additional steps, one ofwhich may be hydrolysis of the phosphoenzyme intermediate. Thesimilarity in the reduction in k_(cat) on mutation of Arg166 to alaninefor the phage enzymes suggests that the same steps may be altered in aquantitatively similar manner in the mutant phage enzyme as in themutant soluble enzyme.

Thus, enzymes displayed on phage show qualitatively similarcharacteristics to soluble enzymes.

Example 31 Demonstration Using Ultrafiltration that Cloned AlkalinePhosphatase Behaves as Part of the Virus Particle

The construct fdphoAla166 (derived in example 11) was converted back tothe wild type residue (arginine) at position 166 by in vitro mutagenesis(Amersham International) using the printer APARG166: 5′TAGCATTTGCGCGAGGTCACA 3′ (SEQ ID NO:51).

This construct with the wild type insert was called fdphoArg166. E. coliTG1 or KS272 cells (cells with a deletion in the endogenous phoA gene,Strauch and Beckwith, 1988 Supra) containing either fd-phoAla166,fdphoArg166 or fd-CAT2 were grown for 16 hours at 37° C. in 2×TY with 15μg/ml tetracycline. Concentrated phage were prepared as follows.Phage-enzyme cultures are clarified by centrifugation (15 min at 10,000rpm, 8×50 ml rotor, sorval RC-5B centrifuge). Phage are precipitated byadding ⅕ volume 20% polyethylene glycol, 2.5 M Nacl, leaving for 1 hr at4° C., and centrifuging (as above). Phage pellets are resuspended in 10mM Tris-HCl, pH 8.0 to 1/100th of the original volume, and residualbacteria and aggregated phage removed by centrifugation for 10 to 15minutes in a bench microcentrifuge at 13000 rpm at 4° C.

SDS/Polyacrylamide gel electrophoresis and western blotting werebasically as described previously (example 2). Denatured samplesconsisting of 16 μl of a 50 fold concentrate of phage were separatedusing a 10% SDS/polyacrylamide gel and detected with polyclonalantiserum raised against either E. coli alkaline phosphatase(Northumbria Biologicals, South Nelson Industrial Estate, Cramlington,Northumberland, NE23 9HL) or against the minor coat protein encoded bygene 3 (from Prof. I. Rasched, Universitat Konstanz, see Stengele et al,1990) at 1 in 1000 dilution. This was followed by incubation withperoxidase-conjugated goat-anti-rabbit immunoglobulin (Sigma 1 in 5000)and detection with the ECL Western blotting system (AmershamInternational).

The presence of fusion proteins was confirmed by western blotting ofproteins from phage particles derived from fd-phoAla166 (phage-enzyme)or fd-CAT2 (vector phage). Detection with antiserum raised against thegene 3 protein reveals a product of apparent relative molecular mass(Mr) of 63,000 in vector phage (FIG. 34 e). Although this is differentfrom the predicted molecular weight based on the amino acid sequence(42,000), the natural product of gene 3 has previously been reported toexhibit reduced mobility during electrophoresis (Stengele et al, 1990).

In the fd-phoAla166 sample the largest band has an apparent Mr of115,000, (FIG. 34). Taking into account the aberrant mobility of thegene 3 portion of the fusion, this is approximately the size expectedfrom fusing with an alkaline phosphatase domain of 47 kD. This analysisalso reveals that a proportion of the Gene3 reactive material in thisphage-enzyme preparation is present at the size of the native gene3product, suggesting that degradation is occurring. In the preparationshown in FIG. 34, approximately 5-10% of the gene 3 fusions are intact.In more recent preparations and in all the preparations used in thisexample and example 32, approximately 30-60% of fusions are full length.

The protein of Mr 115,000 is the major protein observed in Western blotsof phage-enzyme derived from TG1 cells when probed with antiserum raisedagainst E. coli alkaline phosphatase (anti-BAP), confirming theassignment of this band to intact fusion. Further, when phage enzyme isprepared using KS272 cells, which have a deletion in the endogenous phoAgene (Strauch & Beckwith, 1988, supra.) it is also the major band. Thereare additional bands at Mr 95000 and 60000 reactive with anti-BAPantiserum which may indicate degradation of the fusion product.

The anti-BAP antiserum also reacts with material running with the dyefront and with a molecule of Mr 45,000 but evidence suggests that thismaterial is not alkaline phosphatase. This pattern is detected in PEGprecipitated vector phage samples (FIG. 34 c) and is not thereforecontributed by protein expressed from the cloned phoA gene. These bandsare detected in culture supernatants of cells carrying fd-CAT2 but isnot detected in the supernatant of uninfected cells (not shown) and soeither represents cross-reactivity with phage encoded material or with aPEG precipitable cellular component leaked from infected cells (Boeke etal, Mol. Gen. Genet. 186, 185-192 1982). Although the fragment of Mr,45,000 is close to the size of free alkaline phosphatase (47,000), it ispresent in phage preparations from KS272 cells which have a deletion inthe phoA locus. Furthermore its mobility is different from purifiedalkaline phosphatase and they can be distinguished by electrophoresis(FIG. 34 d).

Ultrafiltration was used to confirm that the fusion protein behaved asthough it were part of a larger structure, as would be expected for anenzyme bound to a phage particle. Phage samples (100 μl of a 50 foldconcentrate) were passed through ultrafiltration filters with a nominalmolecular weight limit of 300000 daltons (Ultrafree-MC filters,Millipore) by centrifugation for 5 to 15 minutes at 13,000 r.p.m. in anMSE microcentaur microfuge. Retained material was recovered byresuspending in 100 μl of 10 mM Tris, pH 8.0.

Phage-enzyme or free alkaline phosphatase (83 ng) mixed with vectorphage were passed through filters with a nominal molecular weight limitof 300,000 daltons (ULTRAFREE-MC filters, Millipore). FIG. 35 A againshows that the band of Mr, 115,000 is the major product reactive withanti-BAP antiserum. This and the other minor products reactive withanti-BAP are present in material retained by the ultrafiltrationmembrane. Analysis of retained and flow through fractions of phagepreparations derived from KS272 demonstrates that different molecularspecies are being separated by the ultrafiltration membranes. FIG. 35 bshows the protein of Mr 115,000 is retained by the filter whereas theputative degradation products of Mr 95,000 and 60,000 found in phagepreparations derived from KS272 cells, are not retained.

In mixture of alkaline phosphatase and vector phage FIG. 35B(c-f), freealkaline phosphatase (dimer size of 94,000 daltons) is detected in theflow through as a monomer band with Mr 47,000 on denaturingpolyacrylamide gels (FIG. 35B), while the cross reactive molecule foundin vector phage preparations (Mr 45,000) is in retained on the filter(FIG. 35B). This suggests that the cross reactive molecule is part ofthe phage particle and underlines the fact that the ultrafiltrationmembranes are effecting a separation. Thus the expected fusion band inthis phage-enzyme is present in material retained on ultrafiltrationmembranes demonstrating that it is part of a larger structure as wouldbe expected for viral bound enzyme.

Catalytic activity has been demonstrated on phage particles expressingalkaline phosphatase. Table 7 shows that the wild type alkalinephosphatase gene expressed on phage (fd-phoArg166) has a specificactivity (moles of substrate converted per mole of viral particles) of3,700/min. This is close to the turnover value of 4540/min found forpurified alkaline phosphatase by Malamy and Horecker, Biochemistry 3,1893-1897 1964).

Chaidaroglou et al, 1988 supra have shown that substituting alanine forarginine at the active site (residue 166) leads to a reduction in therate of catalysis. Preparations of phage displaying alkaline phosphatasewith this mutation derived from TG1 and KS272 show reduced specificactivities of 380 and 1400 mol substrate converted/mol phage/minrespectively. Enzyme activity was measured in the retained andflow-through fractions prepared by ultrafiltration, shown in FIG. 35(A)and FIG. 35(B). The bulk of activity from phage-enzyme was retained onthe filters whereas the majority of activity from free enzyme passesthrough. Therefore, the enzyme activity in these fusions behaved aswould be expected for virally associated enzyme (not shown). Little orno catalytic activity is measured in preparations of vector phage fromeither TG1 or KS272 cells (Table 7), indication that the catalyticactivities above are due to phage enzyme and not contamination withbacterial phosphatase. Addition of phage particles to soluble enzymedoes not have a significant effect on activity (Table 7).

Little or no catalytic activity is measured in preparations of vectorphage from either TG1 or KS272

Therefore, both the catalytic and immunochemical activity of alkalinephosphatase have been demonstrated to be due to enzyme which is part ofthe phage particle.

Example 32 Affinity Chromatography of Phage Alkaline Phosphatase

Affinity chromatography, using the specific binding properties ofenzymes has proved to be a very powerful method for their purification.The purification of phage-enzymes by this approach would enable thegenetic material encoding the enzyme to be isolated with the enzymeitself. Thus, mutagenesis of cloned enzymes expressed on the surface offilamentous bacteriophage will lead to a whole population of enzymevariants, from which variants with desired binding properties could beisolated.

Soluble alkaline phosphatase (from calf intestine) has been purified bybinding to immobilised arsenate (a competitive inhibitor), and elutingwith inorganic phosphate, which is a product (and competitive inhibitor)of the enzyme reaction (Brenna, O. et al, Biochem. J. 151 291-296 1975).The applicants have determined that soluble alkaline phosphatase from E.coli is also retained by this matrix (not shown). In this example it isdemonstrated that phage displaying E. coli alkaline phosphatase binds toarsenate-Sepharose and can be specifically eluted.

Arsenate-Sepharose was prepared by coupling 4-(p-aminophenylazo) phenylarsonic acid to tyraminyl-Sepharose according to the method of Breena etal, (1975; supra). Affinity chromatography of phage enzyme fdphoArg166(example 31) was carried out in a disposable chromatography column witha 0.5 ml column volume. Columns were prewashed with 100 volumes ofcolumn buffer (100 mM Tris pH 8.4, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% Tween20, Brenna et al, 1975, supra.) 1 ml of a 40 fold concentrate ofphage-enzyme (in column buffer; prepared as in example 31) was loadedand washed through with 100 volumes of column buffer. Bound phage-enzymewas eluted with 5 mls of column buffer containing 20 mM NaHPO₄. Theeluate and wash fractions were quantitated by dot blotting ontonitrocellulose and comparing with known amounts of phage-enzyme. Theblots were detected using sheep anti-M13 antiserum (gift from M.Hobart), anti-sheep peroxidase (Sigma) and enhanced chemiluminescentsubstrate (Amersham). A range of exposures were taken.

Table 8 shows the results of affinity chromatography of phage displayingalkaline phosphatase on arsenate-Sepharose. In separate experimentsphage particles expressing either mutant (fdphoAla 166; example 11) andor wild type (fdphoArg 166) forms are retained on arsenate-Sepharose andeluted with inorganic phosphate. Approximately 0.5 to 3% of added phageenzyme particles loaded (‘input phage’) were specifically eluted withphosphate (‘output phage’) compared to only 0.05% of vector particles.Arsenate is a competitive inhibitor with K_(i); of 20 μM with respect to4-nitrophenyl phosphate. Phage particles antibodies have previously beenisolated on the basis of interactions with similar affinities (example23). This association is in within the range of a large number ofenzyme-ligand interactions suggesting wide applicability for thisapproach.

Table 8 also shows that the infectivity of phage particles expressingenzyme is reduced with compared with vector phage particles. This makestitration of infectious particles an inappropriate means of quantitatingthe number of phage enzyme particles. For this reason the number ofphage were measured by dot blotting and phage were detected withanti-M13 antiserum as above.

Whereas, overall recovery of catalytic activity may be an importantconsideration in enzyme purification, this is not critical withphage-enzymes. Even if only low levels of phage-enzyme bind to and arespecifically eluted from affinity columns, this will generate cloneswhich can subsequently be grown up in bulk as phage-enzymes or can betransferred to expression vectors yielding soluble products.

Example 33 PCR Assembly of DNA Encoding Fab Fragments of an AntibodyDirected Against Oxazolone

Example 25 showed that genes encoding Fab fragments could be subclonedinto vectors fdCAT2 and pHEN1 and the protein domains displayed on thesurface of phage with retention of binding function. This example showsthat the VHCH and VKCK domains can be amplified separately and thenjoined by a linker allowing the expression of the light chain as ageneIII protein fusion and the VHCH fragment as a soluble molecule. Afunctional Fab fragment is then displayed on phage by association ofthese domains. The assembly process, described in this example, isrequired for display of a library of Fab fragments derived from theimmune repertoire if both heavy and light chain domains are to beencoded within a single vector.

The VHCH1 and VKCK domains of a construct (example 25; construct II inpUC19) derived from antibody NQ10 12.5 directed against2-phenyl-5-oxazolone were amplified using PCR. For cloning into thevector fdCAT2′ the oligonucleotides VH1BACKAPA (example 25) and HuIgG1-4CH1FOR (example 40) were used to amplify the VHCH1 domains. For cloninginto pHEN1 VH1BACKSFH5 (example 25) replaced VH1BACKAPA for thisamplification. For cloning into both vectors the VKCK domains wereamplified using VK2BACK (example 25) and CKNOTFOR (example 40). A linkeroligonucleotide fragment containing the bacteriophage fd gene 8terminator and the fd gene 3 promoter was prepared by amplifying theregion containing them from the vector fdCAT2 by PCR using theoligonucleotides.

VK-TERM-FOR

5′ TGG AGA CTG GGT GAG CTC AAT GTC GGA GTG AGA ATA GAA AGG 3′ (SEQ IDNO:52) (overlapping with VK2BACK [example 14])

and

CH1-TERM-BACK

5′AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA TCT AGC TGATAA ACC GAT ACA ATT AAA GGC 3′ (SEQ ID NO:53) (overlapping with HuIgG1-4CH1-FOR)

Assembly of the Fab fragment from the amplified VHCH1 and VKCK domainsand the linker prepared as above was as described in example 14E exceptthat the primers VH1BACKAPA (when cloning into fdCAT2) or VH1BACKSFH5(when cloning into pHEN1) and CKNOTFOR were used for the finalreamplification, thereby introducing restriction sites for cloning intofdCAT2 (Apa1I-NotI) or pHEN1 (SfiI-Nott) the assembled Fab fragment isshown in FIG. 34. No assembled product was seen in the absence oflinker. An assembled scFv prepared according to example 14 is shown forcomparison.

Phage antibodies were prepared as in example 25 and ELISA was performedwith oxazolone as antigen according to example 6. Results were asexpected for Fab fragments cloned in both fdCAT2 and pHEN1 samples,phage particles bound to oxazolone as detected by a positive ELISAsignal.

Example 34 Construction of a Gene III Deficient Helper Phage

To fully realise the potential of the phagemid cloning system, a helperphage lacking gene III is desirable. Rescue of gene III fusions withsuch a helper phage would result in all the progeny phagemids having agene III fusion on their capsid, since there would be no competitionwith the wild type molecule.

Control over the number of fusion molecules contained on each phage willprovide particularly useful. For example, a gene III deficient helperphage can be used to rescue low affinity antibodies from a naiverepertoire, in which high avidity will be necessary to isolate thosephage bearing the correct antibody specificity. The unmutated helperphage can then be used when higher affinity versions are constructed,thereby reducing the avidity component, and permitting selection purelyon the basis of affinity. This will prove a surprisingly successfulstrategy for isolation and affinity maturation of antibodies from naivelibraries.

The strategy chosen to construct the helper phage was to partiallydelete gene III of M13K07 using exonuclease Bal 31. However, phagelacking gene III protein are non-infective so an E. coli strainexpressing gene III was constructed. Wild type M13 gene III wasPCR-amplified with primers gIIIFUFO and gIIIFUBA, exactly as describedin example 24. The PCR product was digested with Eco RI and Hind III andinserted into Eco RI and Hind III-cut pUC19 (not a phagemid as it lacksthe filamentous phage origin of SS DNA replication) under control of thelac promoter. The plasmid was transformed into E. coli TG1, and theresulting strain called TG1/pUC19gIII. This strain provides gIII proteinin trans to the helper phage.

There is a single unique Bam HI site in M13KO7, which is approximatelyin the centre of gIII. Double-stranded M13K07 DNA was prepared byalkaline lysis and caesium chloride centrifugation (Sambrook et al, etsupra. 1989); twenty μg of DNA was cut with Bam H1, phenol extracted andethanol precipitated then resuspended in 50 μl of Bal 31 buffer (600 mMNaCl, 20 mM Tris-HCl pH 8.0, 12 mM CaCl₂, 12 mM MgCl₂ and 1 mM EDTA) anddigested for 4 minutes with 1 unit of Bal 31 (New England BioLabs). Thistreatment removed approximately 1 Kb of DNA. EGTA was added to 20 mM andthe reaction phenol extracted and ethanol precipitated prior topurification of the truncated genome on an agarose gel. The DNA wasrepaired with klenow enzyme and self-ligated, with T4 DNA ligase (NewEngland BioLabs).

Aliquots of the ligation reaction were transformed into competentTG1/pUC19gIII and plated on SOB medium containing ampicillin at 100μg/ml and kanamycin at 50 μg/ml. Colonies were screened for the presenceof a deletion by PCR with primers gIIIFUBA and KSJ12(CGGAATACCCAAAAGAACTGG) (SEQ ID NO:54).

KSJ 12 anneals to gene VI which is immediately downstream of gIII in thephage genome, so distinguishing gIII on the helper phage from thatresident on the plasmid. Three clones gave truncated PCR productscorresponding to deletions of ca. 200, 400 and 800 bp. These clones werecalled M13K07 gIII Δ Nos 1, 2 and 3 respectively. No clones wereisolated from the earlier Bal 31 time points, suggesting that these arein some way lethal to the host cell. Several clones were isolated fromlater time points, but none of these gave a PCR product, indicating thatthe deletion reaction had gone too far.

M13K07 gIII Δ No.s 1, 2 and 3 were cultured and the resulting helperphage tested for their ability to rescue an antibody gIII fusion (scFvD1.3) by ELISA, exactly as described in example 18. As shown in FIG. 37,only one clone, M13K07 gIII Δ No3 was found to rescue the antibody well;in fact the signal using this helper was greater than that observed withthe parent M13 KO7. M13KO7 gIIIΔ No3 rescued phagemids should have amuch higher density of antibody fusions on their surfaces. That this wasindeed the case was demonstrated when the phage used in this ELISA wereanalysed by Western blotting with anti gIII protein antiserum (FIG. 38).This analysis enables estimation of the amount of gIII fusion proteinversus free gIII protein present on the phage(mid) particles.

Only a minute fraction of the gIII protein on the M13K07-rescuedmaterial is present as an intact fusion (FIGS. 38A-38B). The fusionprotein band is induced by IPTG, so is indisputably that synthesised bythe phagemid. As expected, even when the lac promoter driving gIIIfusion protein synthesis is fully induced (100 μM IPTG), wild type gIIIprotein, at a lower copy number and driven from a far weaker promoter,predominates. This is in contrast to the pattern generated by the sameclone rescued with M13K07 gIIIΔNo3, and the pattern generated by fdCAT2-scFv D1.3. In both of these latter cases, there is no competitionwith wild-type gIII and the fusion protein band is correspondinglystronger.

It is worthy of note that construction of M13K07 gIII Δ No3 wasimmensely inefficient: one clone from 20 μg of starting DNA. Moreover,the yield of gIII helper phage from overnight cultures is extremely lowca. 10⁶ cfu/ml compared with ca. 10¹¹ cfu/ml for the parental phage.Despite this, M13K07 gIII No3 rescues the phagemid as well as theparental phage, as judged by the number of phagemid particles producedafter overnight growth. This indicates that trans replication andpackaging functions of the helper are intact and suggest that its ownreplication is defective. Hence it may be that inactivation of gIII isnormally toxic to the host cell, and that M13K07 gIII A No3 was isolatedbecause of a compensating mutation affecting, for example, replication.Phage fd-tet is unusual in that it tolerates mutations in structuralgenes that are normally lethal to the host cell, since it has areplication defect that slows down accumulation of toxic phage products;M13K07 gIIIΔ No3 may also have such a defect.

M13K07g 111Δ No 3 has been deposited at the National Collection of TypeCultures, 61 Colindale Avenue, London, NW9 6HT, UK (Accession No. NCTC12478). On 28 Jun. 1991, in accordance with the regulations of theBudapest Treaty. It contains a deletion of the M13 genome from bases1979 to 2768 inclusive (see Van Wezenbeek, P. G. M. F. et al., Gene II p129-148, 1980 for the DNA sequence of the M13 genome).

Example 35 Selection of Bacteriophage Expressing scFv Fragments DirectedAgainst Lysozyme from Mixtures According to Affinity Using a PanningProcedure

For isolation of an antibody with a desired high affinity, it isnecessary to be able to select an antibody with only a few fold higheraffinity than the remainder of the population. This will be particularlyimportant when an antibody with insufficient affinity has been isolated,for example, from a repertoire derived from an immunised animal, andrandom mutagenesis is used to prepare derivatives with potentiallyincreased affinity. In this example, mixtures of phage expressingantibodies of different affinities directed against hen egg lysozymewere subjected to a panning procedure. It is demonstrated that phageantibodies give the ability to select for an antibody with a K_(d) of 2nM against one with a K_(d) of 13 nM.

The oligonucleotides used in this example are shown in the list below:

Oligonucleotides

VHBHD13APA: 5′-CAC AGT GCA CAG GTC CAA CTG CAG GAG AGC GGT-3′ (SEQ IDNO:55)

VHFHD13: 5′-CGG TGA CGA GGC TGC CTT GAC CCC-3′ (SEQ ID NO:56)

HD13BLIN: 5′-GGG GTC AGG GCA GCC TCG TCA CCG-3′ (SEQ ID NO:57)

HD13FLIN3: 5′-TGG GCT CTG GGT CAT CTG GAT GTC CGA T-3′ T (SEQ ID NO:58)

VKBHD13: 5′-GAC ATC CAG ATG ACC CAG AGC CCA-3′ (SEQ ID NO:59)

VKFHD13NOT: 5′-GAG TCA TTC TGC GGC CGC ACG TTT GAT TTC CAC CTT GGTCCC-3′ (SEQ ID NO:60)

MURD13SEQ: 5′-GAG GAG ATT TTC CCT GT-3′ (SEQ ID NO:61)

HUMD13SEQ: 5′-TTG GAG CCT TAC CTG GC-3′ (SEQ ID NO:62)

FDPCRFOR: 5′-TAG CCC CCT TAT TAG CGT TTG CCA-3′ (SEQ ID NO:63)

FDPCRBAK: 5′-GCG ATG GGT GTT GTC ATT GTC GGC-3′ (SEQ ID NO:64).

Phage displaying scFv fragments directed against lysozyme were derivedfrom cloned Fab fragments in plasmids.

Heavy and light chain variable regions were amplified by the polymerasechain reaction (PCR) from plasmids containing humanized VH-CH1 or VK-CKinserts suitable for production of Fab fragments (gift of J. Foote). Thedissociation constant, Kd for different combinations of the two plasmidscombined as Fabs, are shown below:

Heavy Chain Plasmid Light Chain Plasmid Kd HuH-1 HuK-3  52 nM HuH-1HuK-4 180 nM HuH-2 HuK-3  13 nM HuH-2 HuK-4 (not determined)Primary PCR

The primary PCR of the variable regions was performed by combining thefollowing:

36.5 μl Water

5 μl PCR buffer (10×)

2 μl dNTP (5 mM)

2.5 μl Back oligo (10 pmoles/μl) (VHBHD13APA or VKBHD13)

2.5 μl Forward oligo (10 pmoles/μl) (VHFHD13 or VKFHD13NOT)

The reaction is decontaminated by UV irradiation to destroy foreign DNAfor 5 minutes, and 1 μl of plasmid DNA added (0.1 μg/μl). The permixture was covered with 2 drops of paraffin oil, and placed on the perblock at 94° C. for 5 minutes before the addition of 0.5 μl of Taq DNApolymerase under the paraffin. The cycling conditions used were 94° C.0.1 min, 40° C. 1 min, 72° C. 1.5 min 17 cycles.

The linker (Gly₄-Ser)₃, was amplified from the anti-phOx(2-phenyloxazol-5-one) clone fd-CAT2-scFv NQ11, using the oligosHD13BLIN and HD13FLIN3, with 0.1 μg of plasmid DNA. The PCR cycling usedwas 94° C. 1 min, 25° C. 1.5 min, for 17 cycles.

Amplified DNA was purified by running the samples on a 2% low meltingpoint agarose gel at 90 mA, excising the appropriate bands andextracting the DNA using the Geneclean II Kit (BIO 101 Inc.) for the VHand VK, or by using Spin-X filter units (Costar) for the linker. A finalvolume of 10 μl was used to resuspend the extracted DNA.

PCR Assembly

Assembly of the four single chain Fv Humanized D1.3 (scFv HuD1.3)constructs was by the process of ‘assembly by overlap extension’ example14.

The following were combined:

34.5 μl Water

5 μl PCR Buffer (10×)

2 μl dNTP (5 mM)

2.5 μl Back oligo (10 pmoles/μl) (VHBHD13APA)

2.5 μl Forward oligo (10 pmoles/μl) (VKFHD13NOT)

Once again, the reaction is decontaminate by UV treatment for 5 minutesbefore the addition of 1 μl of the primary PCR products; VH-1 or VH-2,VK-3 or VK-4, plus the linker DNA. The reaction was covered with 2 dropsof paraffin, and heated at 94° C. for 5 minutes before the addition of0.5 μl of Taq Polymerase. The PCR cycling conditions used were 94° C. 1min, 60° C. 1.5 min, 72° C. 2.5 min for 20 cycles.

The aqueous layer under the paraffin was extracted once with phenol,once with phenol: chloroform, once with ether, ethanol precipitated, andresuspended in 36 μl of water. To this was added, 5 μl of 10× Buffer forNotI, 5 μl 1 mg/ml BSA, and 4 μl (40 U) of NotI (New England Biolabs).The restriction was incubated at 37° C. overnight.

The DNA was ethanol precipitated and resuspended in 36 μl of water, and5 μl 10×NEB Buffer 4, 5 μl 1 mg/ml BSA, and 2 μl (40 U) of ApaLI (NewEngland Biolabs). This was incubated at 37° C. for 5 hours; a further 2μl of ApaLI was added and the reaction incubated at 37° C. overnight.

The cut DNA was extracted by gel purification on a 1.3% low meltingpoint agarose gel followed by treatment with Geneclean, to yield theinsert DNA for cloning.

Vector fd CAT2 (prepared and digested with ApaLI and NotI as in example20) and the scFv DNA were ligated as in example 20.

Analysis of Clones

Colonies from the ligations were first screened for inserts by PCRscreening. The PCR mixture was prepared in bulk by combining 14.8 μL1×PCR Buffer, 1 μl dNTP (5 mM), 1 μl Back oligo (FDPCRBAK), 1 μl Forwardoligo (FDPCRFOR), and 0.2 μl Taq polymerase per colony screened. 20 μlof this PCR mixture was aliquoted into a 96 well Techne plate. The topof a colony was touched with a toothpick and twirled quickly into thePCR mixture and the colony rescued by placing the toothpick in aCellwell plate (Nunc) containing 250 μl of 2×TY medium. The PCR mixtureis covered with 1 drop of paraffin and the plate placed on the block at94° C. for 10 minutes before cycling at 94° C. 1 minute, 60° C. 1minute, 72° C. 2.5 minutes.

The clones thus derived were named as below. The affinity of scFvfragments derived the Fab fragments was not determined but previousresults suggests that these are closely related although not necessarilyidentical (R. E. Bird & B. W. Walker TIBTECH 9 132-137, 1991).

Con- struct Name Affinity (Kd) Composition of Fab TPB1VH-HuH2-(Gly₄-Ser)₃-VK-HuK3  13 nM (SEQ ID NO:269) TPB2VH-HuH1-(Gly₄-Ser)₃-VK-HuK4 180 Nm (SEQ ID NO:270) TPB3VH-HuH2-(Gly₄-Ser)₃-VK-HuK4 (Unknown)(SEQ ID NO:271) TPB4VH-HuH1-(Gly₄-Ser)₃-VK-HuK3  52 nM (SEQ ID NO:272)

Preparation of phage and ELISA was as described in example 6. The clonesgenerated in fd CAT2 were shown to bind lysozyme as expected.

Affinity Selection

Selection of Highest Affinity Binding Phage

Mixing experiments were performed in which fd-CAT2 scFvD1.3 phage(example 19) were mixed with either fd-CAT2 TPB1, fd-CAT2 TPB2, orfd-CAT2 TKPB4, and used in one round of panning.

The general method used for affinity selection by panning is thatdetailed below. Any deviation from this protocol is described at therelevant point. Panning plates were placed on a rocking platform betweenmanipulations.

Falcon 35 mm Tissue Culture dishes were coated overnight with 1 ml ofLysozyme (various concentrations) dissolved in 50 mM Sodium HydrogenCarbonate, pH 9.6, and blocked with 2 ml 2% MPBS at room temperature for2 hours. Phage were prepared in 1 ml 2% MPBS and rocked at roomtemperature for 2 hours. Plates were washed for 5 minutes with 2 ml ofthe following solutions; 5 times with PBS, PBS-Tween, 50 mM Tris-HCl, pH7.5; 500 mM Sodium Chloride, 50 mM Tris-HCl, pH 8.5; 500 mM SodiumChloride, 50 mM Tris-HCl, pH 9.5; 500 mM Sodium Chloride, 50 mM SodiumHydrogen Carbonated, pH 9.6; 500 mM Sodium Chloride. Phage were theneluted by adding 1 ml 100 mM Triethylamine and rocking for 5 minutesbefore removing the eluate which was neutralised with 100 μl 1.0 MTris-HCl, pH 7.4.

Plates were coated overnight with Lysozyme at the concentration listedbelow.

Colonies from the single round of panning were probed with eitherMURDSEQ (for fdCAT2 scFvD1.3) or HUMD13SEQ (for fdCAT2 TPB constructs).

Circles of nitrocellulose (Schleicher & Schuell, BA 85, 0.45 μm) werelabelled in pencil and lowered gently onto the colonies derived from thepanning experiments and left for one minute. The filters were thenpulled off quickly from one edge and placed colony side up on a piece of3 mM paper (Whatman) soaked in Denaturing solution (500 mM SodiumHydroxide; 1.5 M Sodium Chloride) for 5 minutes. They were thentransferred to 3 mM soaked in Neutralizing Solution (3.0 M SodiumChloride; 500 mM Tris-HCl, pH 7.5) for 1 minute, and then to 3 mM soakedin 5×SSC; 250 mM Ammonium Acetate for 1 minute. The filters were thenair dried before baking in an 80° C. vacuum oven for 30 minutes.

The oligonucleotide probe was prepared by combining the following:

2 μl oligonucleotide (1 pmoles/μl)

2 μl γ-32P ATP (3000 Ci/mmole) (Amersham International plc)

2 μl 10× Kinase buffer (0.5 M Tris-HCl, pH 7.5; 100 mM MagnesiumChloride; 10 mM DTT)

12 μl Water

2 μl Polynucleotide Kinase (20 Units)

This was incubated at 37° C. for 1 hour.

Hybridization was performed in the Techne HB-1 Hybridiser. The bakedfilters were pre-hybridized at 37° C. in 40 ml of Hybridization Buffer(10 ml 100 mM Sodium pyrophosphate; 180 ml 5.0 M Sodium chloride;

-   20 ml 50×Denharts Solution; 90 ml 1.0 M Tris-HCl, pH 7.5; 24 ml 250    mM EDTA; 50 ml 10% NP40; made to 1 liter with water; 60.3 mg rATP;    200 mg yeast RNA (Sigma)), for 15 minutes before the addition of the    μl of the kinased oligo. The filters were incubated at 37° C. for at    least one hour, and then washed 3 times with 50 ml of 6×SSC at    37° C. for 10 minutes (low stringency wash). Filters were air dried,    covered with Saran wrap and exposed overnight with Kodak X-AR film.    Selection of fd-CAT2 scFv D1.3 from fd-CAT2 TPB4

FIG. 39, summarizes the results from panning experiments using a mixtureof the high affinity fd-CAT2 scFv D1.3 phage (Kd-2 nM) and the fd-CAT2TPB4 construct (Kd-52 nM).

At a coating concentration of 3000 μg/ml Lysozyme, little or noenrichment could be obtained. It was however, possible to get enrichmentfor the scFv D1.3 phage when a lower concentration of Lysozyme was usedfor coating the plates. The best enrichment value obtained was from 1.5%fd-CAT2 scFv D1.3 in the starting mixture, to 33% fd-CAT2 scFv D1.3 inthe eluted faction, on a plate coated overnight with 30 μg/ml Lysozyme.

Selection of fd-CAT2 scFv D1.3 from fd-CAT2 TPB1

Enrichment for the high affinity scFv D1.3 phage over the fd-CAT2 TPB1phage (Kd-13) nM, could only be shown from experiments where the plateshad been coated overnight with low concentrations of Lysozyme, as shownin FIG. 40.

In summary, single chain Fv versions of a series of humanized D1.3antibodies have been constructed in phage fd-CAT2. By affinity selectionof fd-CAT2 phage mixtures, by panning in small petri dishes, it wasshown that the high affinity scFv D1.3 phage, could be preferentiallyselected for against a background of lower affinity scFv HuD1.3 phage.

Example 36 Expression of Catalytically Active Staphylococcal Nuclease onthe Surface of Bacteriophage fd

Examples 11 and 12 showed that alkaline phosphatase from E. coli can beexpressed as a catalytically active enzyme on the surface ofbacteriophage fd. Here we show that Staphylococcal nuclease can also beexpressed in a catalytically active form suggesting that thismethodology may be general.

The gene for the enzyme Staphylococcal nuclease (SNase) was amplifiedfrom M13 mp18-SNase (Neuberger, M. S. et al Nature 312 604-608, 1984) byPCR using primers with internal ApaLI(5′-GGAATTCGTGCACAGAGTGCAACTTCAACTAAAAAATTAC-3′) (SEQ ID NO:65) and NotI(5′-GGGATCCGCGGCCGCTTGACCTGAATCAGCGTTGTCTTCG-3′) (SEQ ID NO:66)restriction sites, cloned into phage vector fd-CAT2 after digestion withApaLI-NotI restriction enzymes and the nucleotide sequence of the SNasegene and junctions with gene III checked by DNA sequencing. Thefd-tet-SNase phage was prepared from the supernatant of infected E. coliTG1 cultures by three rounds of PEG precipitation, and the fusionprotein demonstrated by SDS-gel electrophoresis and Western blottingusing rabbit anti-g3p antiserum (Prof I. Rasched, Konstanz) andperoxidase-labelled goat anti-rabbit antibodies (Sigma) (FIG. 41) asdescribed in example 27. As well as the fusion protein band (calculatedMr 59749, but runs at a higher position due to the aberrant g3pbehaviour), a smaller (proteolytic ?) product is seen.

The fusion protein was shown to be catalytically active by incubation ofthe fd-tet-SNase phage (4×10⁹ tetracyclin resistant colonies [TU]) withsingle stranded DNA (1 μg) for 1 hr at 37° C. in the presence of Ca₂+,and analysis of the digest by agarose gel electrophoresis (FIG. 42).Nuclease activity was not detected with the parent fd-CAT2 (2×10¹⁰ TU)phage alone or after three rounds of PEG precipitation of mixtures offd-CAT2)(2×10¹⁰ TU) with SNase (0.7 μg). Thus the nuclease activityresults from the display of the enzyme on the surface of the phage andnot from co-precipitated or soluble SNase set free by degradation of thefusion protein. The nuclease activity of fd-tet-SNase (FIG. 42) lies inthe same order of magnitude, (2×10⁸ TU and assuming three copies ofSNase per TU) as an equimolar amount of SNase (0.03 ng or 10⁹particles), and like the authentic SNase was dependent on Ca²+, sinceincubation with 40 mM MgCl² and 25 mM EGTA blocked activity (not shown).

Example 37 Display of the Two Aminoterminal Domains of Human CD4 on theSurface of fd Phage

The protein CD4, a member of the immunoglobulin superfamily, is a cellsurface receptor involved in MHC class II restricted immune recognition.It is also recognised by the protein gp120 derived from the humanimmunodeficiency virus (AIDS virus). The first two domains (named V1 andV2, residues 1-178) of the surface antigen CD4 were amplified frompUC13-T4 (gift from T. Simon) containing the human cDNA of CD4, by PCRusing primers with internal ApaLI (5′-GGA ATT CGT GCA CAG AAG AAA GTGGTG CTG GGC AAA AAA GGG G-3′) (SEQ ID NO:67) and NotI (5′-GGG ATC CGCGGC CGC AGC TAG CAC CAC GAT GTC TAT TTT GAA CTC-3′) (SEQ ID NO:68)restriction sites. After digestion with these two enzymes, thePCR-product was cloned into fdCAT2, and the complete nucleotide sequenceof the CD4-V1V2 DNA and junctions with gene III checked by dideoxysequencing using oligonucleotides fd-seq1 (5′-GAA TTT TCT GTA TGA GG)(SEQ ID NO:69), CD4-seq1 (5′-GAA GTT TCC TTG GTC CC-3′) (SEQ ID NO:70)and CD4-seq2 (5′-ACT ACC AGG GGG GCT CT-3′) (SEQ ID NO:71). In the sameway, a fd-CD4-V1 version was made, linking residues 1-107 to theN-terminus of gene III, using previously mentioned primers andoligonucleotide 5′-GGG ATC CGC GGC CGC GGT GTC AGA GTT GGC AGT CAA TCCGAA CAC-3′ (SEQ ID NO:72) for amplification, PCR conditions and cloningwere essentially as described in example 15 except that digestion waswith ApaLI and NotI (used according to the manufacturers instructions).

Both fd-CD4-V1 and fd-CD4-V1V2 phages were prepared from the supernatantof infected E. coli TG1 cultures by three rounds of PEG precipitation,thereby concentrating the sample 100-fold for ELISA analysis. The fusionprotein was detected in a Western blot (results not shown) with a rabbitanti-gene III antiserum, and revealed bands of the expected size.

Binding of the CD4 moiety to soluble gp120 (recombinant HIV-IIIB gp120from CHO cells, ADP604, obtained from the Aids Directed Programme,National Institute for Biological Standards and Controls, South Mimms,Potters Bar, UK) was analysed in an ELISA, using 5 μg/ml gp120 forcoating (overnight, in PBS). Anti-M13 antiserum was used to detect boundphage; all other conditions were as in Example 9.

FIG. 43 shows the ELISA signals of wild-type phage (fd-tet) and bothCD4-phages. Both CD4-phages can bind gp120, but fd-CD4-V1V2 binds muchstronger to gp120 than fd-CD4-V1. The binding competitors, soluble CD4(recombinant soluble CD4 from Baculovirus, ADP 608; from the AIDSDirected Programme) (25 μg/ml) or soluble gp120 (20 μg/ml), addedtogether with the 50 μl phage stock sample during the ELISA, decreasedthe signal to background level. These results indicate that phagebinding to gp120 is mediated by the CD4 molecule displayed at itssurface, and that binding is stronger when the two aminoterminal domainsof CD4 are presented.

Thus, CD4 is a cell surface receptor molecule which is active whendisplayed on bacteriophage fd. Like the PDGF-BB receptor, the functionaldisplay of which is described in examples 15 and 16, CD4 is a member ofthe immunoglobulin superfamily and this result suggests that this classof molecule may be generally suitable for display on the surface ofphage.

Example 38 Generation and Selection of Mutants of anAnti-4-Hydroxy-3-Nitrophenylacetic Acid (NP) Antibody Expressed on PhageUsing Mutator Strains

It will sometimes be desirable to increase the diversity of a pool ofgenes cloned in phage, for example a pool of antibody genes, or toproduce a large number of variants of a single cloned gene. There aremany suitable in vitro mutagenesis methods. However, an attractivemethod, particularly for making a more diverse population of a libraryof antibody genes, is to use mutator strains. This has the advantage ofgenerating very large numbers of mutants, essentially limited only bythe number of phage that can be handled. The phage display system allowsfull advantage to be taken of this number to isolate improved or alteredclones.

Nucleotide sequences encoding an antibody scFv fragment directed against4-hydroxy-3-nitrophenylacetic acid (NP), scFvB18, derived as in example14 from a monoclonal antibody against NP were cloned into fdCAT2 usingApaLI and NotI restriction sites as in example 11 to createfdCAT2scFvB18 or into fdDOGKan (fdCAT2 with its tetracycline resistancegene removed and replaced by a kanamycin resistance gene) using PstI andNotI restriction sites to create fdDOGKanscFvB18 or into the phagemidvector pHEN1 using the restriction sites SfiI and NotI as a fusionprotein with gene III to create pHEN1scFvB18.

The following mutator strains (R. M. Schaaper & R. L. Dunn J. Mol. Biol.262 1627-16270, 1987; R. M. Schaaper Proc. Natl. Acad. Sci. U.S.A. 858126-8130 1988) were used:

NR9232: ara, thi, mutD5-zaf13::Tn10, prolac, F′prolac

NR9670: ara, thi, azi, mutT1, leu::Tn10, prolac

NR9292: ara, thi, mutH101, prolac, F′prolac

NR9084: ara, thi, mutT1, azi, prolac, F′prolacI⁻Z⁻ ΔM15 M15

NR9046: ara, thi, supE, rif, nalA, metB, argE(am), prolac, F′prolac werekind gifts of Dr. R. M. Schaaper (Department of Health & Human Services,N1H, PO Box 12233, Research Triangle Park, N.C. 27709)

NR9046mutD5: NR9046 mutD5::Tn10

NR9046mutT1: NR9046 mutT1::Tn10

were constructed by P1 transduction according to standard procedures.Mutator strains were transfected with fdCAT2scFv818 of fdDOGKanscFvB18and transfectants selected for antibiotic resistance. Transfectants weregrown for 24 h at 37° C. before mutant phage was harvested by PEGprecipitation. The mutant phage were selected on a 1 ml NIP(4-hydroxy-3-iodo-5-nitrophenylacetic acid)-BSA-Sepharose affinitycolumn (prepared according to the manufacturers instructions) prewashedwith 200 ml of PBS and blocked by 20 ml MPBS. Phage were loaded on thecolumn in 10 ml MPBS and unbound material reapplied to ensure completebinding. The column was subsequently washed with 10 ml of MPBS and 500ml of PBS. Phage bound to the affinity matrix was eluted with 5 columnvolumes of 0.33 mM NIP-Cap (example 48).

Phage eluate was incubated for 30 min to 1 h with log phase (2×10⁸cells/ml) E. coli mutator strains without antibiotic selection. Theinfected cells were then diluted 1:100 in 2×TY and grown for 24 h withantibiotic selection (15 μg/ml tetracyclin or 30 μg/ml kanamycin forfdCAT2scFvB18 or fdDOGKanscFvB18 respectively). Phage from this culturewas used for another round of affinity selection and mutation.

Binding of phage antibodies was assayed by ELISA as in example 9 exceptthat ELISA plates were coated with NIP-BSA(4-hydroxy-3-iodo-5-nitrophenylacetyl-BSA; 0.4 mg/ml). Culturesupernatants were prepared following growth in Cellwells as described inexample 21 and 20 μl of culture supernatant was added to each welldiluted to 200 μl with MPBS.

Phage samples giving signals in ELISA of more than twice the backgroundwere tested ELISA as above for non-specific binding against lysozyme,BSA or Ox-BSA (example 9). Specificity for NIP was further confirmed byan ELISA in which serial dilutions of NIP-CAP were added together withphage antibodies. Addition of increasing concentrations of NIP-CAPreduced the ELISA signal to the background level.

Phage giving positive signals in ELISA were sequenced and 2 differentmutants were subcloned into pHEN1 phagemid and transformed into HB2151for soluble expression and TG1 for phage display (example 27).

For expression of soluble scFv fragments, transformants in E. coliHB2151 were grown at 37° C. in 1 liter 2×TY, 0.2% glucose, 0.1 mg/mlampicillin to an OD600 of 1 and expression of soluble scFv fragmentsinduced by adding IPTG to 1 mM. Cultures were shaken at 30° C. for 16 h.

Soluble scFvB18 was concentrated from crude bacterial supernatant in aFLOWGEN ultrafiltration unit to a volume of 200 ml.

The concentrate was passed two times over a 2 ml column ofNIP-BSA-Sepharose prewashed with 200 ml of PBS. The column was washedwith 500 ml of PBS and 200 ml of 0.1M Tris pH7.5, 0.5M NaCl and phageantibodies eluted with 50 mM Citrate buffer pH2.3. The eluate wasimmediately neutralised with 1MTris pH8. The eluate was dialysed againsttwo changes of 1 liter PBS, 0.2 mM EDTA, Precipitated protein wasremoved by centrifugation at 10000 g and protein yield was determined bymeasuring the absorbance at 280 nm of the supernatant.

After 4 rounds of mutation and selection, isolated clones were screenedand in one or two rare examples strongly positive ELISA signals wereobtained from phage antibodies derived from the mutation of each offdCAT2scFvB18 and fdDOGKanscFvB18 in the ELISA. The ELISA conditionswere such that the parent phage fdCAT2scFvB18 only generated weaksignals. These phage antibodies giving strongly positive ELISA signalswere enriched in further rounds by a factor of roughly 2.5 per round.Forty phage antibodies giving strongly positive signals were sequencedand they each displayed single mutations in six different positions inthe scFvB18 nucleotide sequences, five of which reside in the lightchain. More than 70% of the mutations occurred at positions 724 and 725changing the first glycine in the J segment of the light chain(framework 4) to serine (in 21 cases) or aspartate (in 3 cases). Themutations found are shown in Table 9. The sequence of scFvB18 is shownin FIG. 44( i) through 44(ii).

The nucleotide sequences encoding the scFv fragments of a frameworkmutant with the above glycine to serine mutation, as well as a mutantwhere Tyr in the CDR3 of the light chain had been mutated to aspartate,were amplified by PCR from the phage antibody clones and subcloned intopHEN1 phagemid (essentially as in example 25). This avoids possibleproblems with geneIII mutations caused by the mutator strains. The samepattern of ELISA signals was seen when the mutants were displayed onphage following rescue of the phagemid with helper phage (as describedin example 25) as when the mutants were assayed when expressed from thephage genome as above.

The scFv fragments from scFvB18 and the scFv fragments containing theglycine to serine and tyrosine to aspartate mutations respectively wereexpressed in solution (following transformation into E. coli HB2151 asin example 27) at 30° C. They showed no differences in the ELISA signalsbetween wild-type B18 and the framework mutant. The signal obtained fromthe phage antibody with the Tyr mutated to aspartate in CDR3 of scFvB18was about 10× stronger. Expression yields were found to be comparable asjudged by Western blotting using an antiserum raised against g3p (asdescribed above). Affinity measurements were performed usingfluorescence quenching as described in example 23. Affinity measurementof affinity purified scFv fragments however showed scFvB18, and thescFvB18 (Gly→Ser) and scFvB18(Tyr→Asp) mutants all to have a comparableaffinity of 20 nM for NIP-CAP.

A Western blot using an anti-geneIII antibody showed the frameworkmutant had suffered significantly less proteolytic cleavage thanscFvB18.

Hence, the use of mutator strains generates a diverse range of mutantsin phage antibodies when they are used as hosts for clones for gene IIIfusions. In this case some of the clones exhibit higher ELISA signalsprobably due to increased stability to proteolytic attack. The mutatorstrains can therefore be used to introduce diversity into a clone orpopulation of clones. This diversity should generate clones withdesirable characteristics such as a higher affinity or specificity. Suchclones may then be selected following display of the proteins on phage.

Example 39 Expression of a Fv Fragment on the Surface of Bacteriophageby Non-Covalent Association of VH and VL Domains

This example shows that functional Fv fragments can be expressed on thesurface of bacteriophage by non-covalent association of VH and VLdomains. One chain is expressed as a gene III fusion and the other as asoluble polypeptide. Thus Fv fragments can be used for all thestrategies discussed for Fab fragments including dual combinatoriallibraries (example 26).

A useful genetic selection system for stably associated Fv fragmentscould be established if the expression of Fv fragments as fusionproteins on the phage surface would be possible such that one V domainis fused to the gene III protein and the other V domain is expressedseparately in secreted form, allowing it to associate with the V domainon the fusion protein provided the interaction strength is sufficientlyhigh. This idea was tested in a model experiment using the V domainsfrom the anti-hen egg lysozyme antibody D1.3 by fusing the D1.3 VK geneto gene III and separately expressing the D1.3 VH domain.

Experimentally this was achieved as follows: The vector fd-DOG1 wasdigested with the restriction enzymes PstI and Xho1. From the Fvexpression plasmid pSW1-VHD1.3-VKD1.3myc version 3/pUC119 (Ward et al.,1989 supra) a Pst 1/Xho I-digested restriction fragment was isolatedthat carries the VH domain coding sequence (terminated by 2 stopcodons), a spacer region between VH and VK genes including aribosome-binding site for expression of the VK gene, a pelB leadersequence, and, following in frame, the VK gene. This fragment was clonedinto the digested fd-DOG vector to generate the construct fd-tet FvD1.3. As shown on the map in FIG. 45, the dicistronic VH/VK-gene IIIoperon is transcribed from the gene III promoter; secretion of the VHdomain is achieved by the gene III protein leader, secretion of theVK-geneIII fusion protein by the pelB leader sequence. For controlpurposes a second construct with the name fd-tet Fv D1.3 (ΔS-Stuffer)was made by a similar route as described above: the VH used in thisconstruct carries an insertion of a 200 by fragment in the Sty Irestriction site at the junction of VH CDR 3/FR4, thus interrupting theVH with several in frame stop codons. It is known from previous workthat this insertion sufficiently disrupts the VH structure to abolishbinding to the antigen lysozyme when expressed either as a soluble Fv orsingle-chain Fv fragment or as a single-chain Fv fragment on phagesurface. This construct was used as a control. TG1 bacteria carryingeither the fd-tet Fv D1.3, fd-tet Fv D1.3 (ΔS-Stuffer) or assingle-chain wild-type control fd-tet scFv D1.3 plasmids were grown inliquid culture (medium 2×TY containing 15 μg/ml tetracycline) for 24 hto produce phage particles in the supernatant. After removal ofbacterial cells by centrifugation the phage titer in the supernatantswas determined by re-infecting exponentially growing TG1 cells withdilutions of the supernatants and scoring tetracycline-resistantcolonies after plating on tetracycline-plates. The infectious phagetiters achieved were 1×10¹¹ tetR transducing units/ml for thesingle-chain wild-type control fd-tet scFv D1.3 and 2×10¹⁰ tetRtransducing units/ml for Fv phage constructs fd-tet Fv D1.3 and fd-tetFv D1.3 (ΔS-Stuffer).

ELISA of Hen Egg Lysozyme was Performed as in Example 2.

The results are shown in FIG. 46. Phage derived from bacteria carryingand expressing the Fv construct fd-tet Fv D1.3 bind to the immobilisedhen egg lysozyme, and when taking the phage titer into account, indeedapparently better than the single-chain Fv bearing phages produced byfd-tet scFv D1.3 carrying bacteria. The specificity of the reaction andthe requirement for a functional VH domain is demonstrated by the fd-tetFv D1.3 (AS-Stuffer) control in which disruption of the VH domain andconsequently of the Fv fragment association eliminates binding tolysozyme.

As a final control of the expected structure of the VK/geneIII fusionprotein a Western Blot was carried out. 20 μl of phage suspensionsconcentrated 100 fold by two sequential precipitations with PEG wereapplied to a 10% SDS-PAGE gel, electrophoretically separated and thentransferred to a PVDF membrane (Immobilon, Millipore) in a semi-dryWestern transfer apparatus (Hoefer). Remaining binding sites on thefilter were blocked by 1 h incubation with 3% BSA in PBS, and detectionof the gene III protein accomplished by incubation with a 1:1000 dilutedrabbit anti-geneIII antiserum for 2 h, several washes in PBS/0.1% Tween20, incubation with peroxidase-conjugated goat anti-rat immunoglobulinantibodies, washes and development with the chromogenic substratediaminobenzidine/CoCl₂/0.03% H₂O₂. The Fv phage fd-tet Fv D1.3 yields aband for the gene III fusion protein (data not shown), that isintermediate in size between the bands obtained for a wild-type gene IIIprotein from fd-DOG1 and the scFv-gene III fusion protein from fd-tetscFv D1.3, thus proving the presence of a single immunoglobulin domaincovalently fused to the gene III product in the Fv phage.

In summary, Fv-gene III fusions in which one V domain is fused to thegene III protein and the other V domain associates non-covalently can bepresented in functionally active form on the surface of filamentousphage. This opens the possibility to genetically select for stablyassociated Fv fragments with defined binding specificities from V genelibraries expressed in phages.

Example 40 A PCR Based Technique for One Step Cloning of Human V-Genesas Fab Constructs

This example describes a PCR based technique to “assemble” human Fabs bysplicing together the heavy and light chain DNA with a separate piece of‘linker’ DNA. A mixture of universal primers is used which should makethe technique applicable to all human V-genes.

The general technique for PCR assembly of human V-genes to create a Fabconstruct is described. The efficiency of this technique was assessed by“assembling”, cloning and expressing a human anti rhesus-D (Rh-D) Fabfrom a IgG-K monoclonal hybridoma. We also demonstrate the potential torescue human monoclonal antibodies from polyclonal cell populations byassembling, cloning, expressing and isolating an IgG-lambda monoclonalanti-Rh-D Fab from a polyclonal lymphoblastic cell line (LCL).

The overall strategy for the PCR assembly is shown in FIG. 47 and isdescribed in more detail below. For Fab assembly, the VH-CH1 and VK-CKor V lambda-C lambda light chains are amplified from first strand cDNAand gel purified. Heavy and light chain DNA are then combined togetherwith linker DNA and flanking oligonucleotides in a new PCR reaction.This results in a full length Fab construct since the 5′ end of thelinker DNA is complementary to the 3′ end of the CH1 domain and the 3′end of the linker is complementary to the 5′ end of the light chaindomain. The linker DNA contains terminal residues of the human CH1domain, the bacterial leader sequence (pelB) for the light chain and theinitial residues of the VK or V lambda light chain. Finally, after gelpurification, the Fab construct is reamplified with flankingoligonucleotides containing restriction sites for cloning.

Oligonucleotide primers: In order to develop the PCR cloning of human Vgenes it was necessary to design a new range of human specificoligonucleotide primers.

The PCR primers at the 5′ end of the VH and VK and Vlambda gene exon(BACK primers) are based on sequence data extracted from the Kabatdatabase, (Kabat, E. A. et al, Sequences of Proteins of ImmunologicalInterest. 4th Edition. US Department of Health and Human Services. 1987)the EMBL database, the literature (Chuchana, P., et al, Eur J. Immunol.1990. 20:1317) and unpublished data. The sequence of the VH, VK andVlambda primers are given in table 1. In addition, extended VH primerswith SfiI sites at the 5′ end were also designed (Table 10) for adding arestriction site after assembly.

Table 10 also shows the 3′ primers (FORWARD primers) designed for thePCR based cloning of human V genes. There are two sets of thesedepending on whether a Fab or scFv is to be produced. For Fab assembly,the forward primer was based at the 3′ end of the CH1 domain, CK domainand Clambda domain. In addition, the CK and C2 FORWARD primers were alsosynthesized as extended versions with NotI sites at their 5′ ends.

Primers complementary to the CH₁ forward primers and the VkK and Vlambda back primers were synthesized to permit generation of linker DNAby PCR amplification of a plasmid template containing the Fab linker(Table 10). To ensure adequate amplification, the primers were extendedinto the actual linker sequence.

A RNA Preparation

This is essentially the same as described in Example 14, but usingmaterial of human origin. In the results given in this example humanhybridoma and human polyclonal lymphoblastic cell lines were used.

B cDNA Preparation

Approximately 4 μg of total RNA in 20 μl water was heated at 65° C. for3 minutes, quenched on ice and added to a 30 ul reaction mixtureresulting in a 50 ul reaction mixture containing 140 mM KCl, 50 mM Tris,HCl (pH8.1 @ 42° C.), 8 mM MgCl₂, 10 mM DTT, 500 uM deoxythymidinetriphosphate 500 uM deoxycytosine triphosphate, 500 uM deoxyadenosinetriphosphate and 500 uM deoxyguanosine triphosphate, 80 units of humanplacental RNAse inhibitor and 10 pmol of the appropriate Forward primer(HulgG1-4-CH1FOR, HuIgMFOR, HuCKFOR, HuCLFOR). Two ul (50 units) ofavian myeloblastosis virus (AMV) reverse transcriptase was added, thereaction incubated at 42° C. for 1 hour, heated to 100° C. for 3minutes, quenched on ice and centrifuged for 5 minutes.

C Primary PCRs

For the primary PCR amplifications, an equimolar mixture of theappropriate family based BACK and FORWARD primers was used. (Seespecific examples 40a and 40b given later in this example). A 50 ulreaction mixture was prepared containing 5 ul of the supernatant fromthe cDNA synthesis, 20 μmol total concentration of the FORWARD primers,250 uM dNTPs, 50 mM KCl, 100 mM Tris. HCl (pH 8.3), 1.5 mM MgCl₂, 175ug/ml BSA and 1 ul (5 units) Thermus aquaticus (Taq) DNA polymerase(Cetus, Emeryville, Calif.). The reaction mixture was overlaid withparaffin oil and subjected to 30 cycles of amplification using a Technethermal cycler. The cycle was 94° C. for 1 minute (denaturation), 57° C.for 1 minute (annealing) and 72° C. for 1 minute (extension). Theproduct was analyzed by running 5 ul on a 2% agarose gel. The remainderwas extracted twice with ether, twice with phenol/chloroform, ethanolprecipitated and resuspended in 50 ul of H₂O.

D Preparation of Linker

To make the Fab linker DNA, 13 separate PCR reactions were performedusing HulgG1-4-CH1FOR and each of the reverse VK or V lambdaoligonucleotides. The template was approximately ing of pJM-1Fab D1.3(FIG. 48) The PCR reaction reagents were as described above and thecycle was 94°: 1 min, 45°: 1 min and 72°: 1 min. The linkers wereanalyzed on a 4% agarose gel, purified on a 2% agarose gel, eluted fromthe gel on a Spin-X column and ethanol precipitated.

E Assembly PCRs

For PCR assembly of a human Fab approximately 1 ug of a primary heavychain amplification and 1 ug of a primary light chain amplification weremixed with approximately 250 ng of the appropriate linker DNA in a PCRreaction mixture without primers and cycled 7 times (94°: 2 min, 72°:2.5 min) to join the fragments. The reaction mixture was then amplifiedfor 25 cycles (94°: 1 ml, 68°-72°: 1 min, 72°: 2.5 min) after theaddition of 20 μmol of the appropriate flanking BACK and FORWARDprimers.

F Adding Restriction Sites

The assembled products were gel purified and reamplified for 25 cycles(94°: 1 min, 55°: 1 min, 72°: 25 min) with the flanking oligonucleotidescontaining the appended restriction sites. PCR buffers and NTPs were asdescribed previously.

Specific Examples of PCR Assembly of Human Immunoglobulin Genes

a. PCR assembly of a Fab from a human hybridoma: the human monoclonalanti Rh-D cell lines Fog-1 (IgG-k) was derived from EBV transformationof the PBLs of a Rh-D negative blood donor immunized with Rh-D positiveblood and has been previously described (Melamed, M. D., et al., J.Immunological Methods. 1987. 104:245) (Hughes-Jones N. C., et al.,Biochem. J. 1990. 268:135) (Gorick, B. D. et al., Vox. Sang. 1988.55:165) Total RNA was prepared from approximately 10⁷ hybridoma cells.First strand cDNA synthesis was performed as described above using theprimers HulgG1-4-CH1FOR and HuCKFOR. Primary PCRs were performed for theVH-CH₁ using a mixture of the 6 HuVHBACK primers and HuIgG1-4CG1FOR andfor the VK-CK using a mixture of the 6 HuVKBACK primers and HuCKFOR. AFab construct was assembled as described above, restricted with SfiI andNotI, gel purified and ligated into pJM-1Fab D1.3 restricted with SfiIand NotI. The ligation mixture was used to transform competent E. coliE.M.G. cells. Ninety-six clones were toothpicked into media inmicrotitre plate wells, grown to mid-log phase at 30° C. and thenexpression of the Fab was induced by heat shocking at 42° C. for 30 minfollowed by growing for 4 hours at 37° C. The ninety-six clones werethen screened for anti-Rh-D activity as described below.b. assembly of human Fabs from a polyclonal (LCL): A polyclonal LCL “OG”was derived from EBV transformation of approximately 10⁷ peripheralblood lymphocytes (PBLs) from a Rh-D negative donor immunized with Rh-Dpositive red blood cells. The cells were plated at a concentration ofapproximately 10⁵ cells per well. Positive wells were identified byscreening the cells harvested and then subcloned once. Typing of thewell indicated that an IgG-lambda antibody was being produced. At thisstage, total RNA was prepared from approximately 10⁶ cells. First strandcDNA synthesis was performed as described above using the primersHulgG1-4CG1FOR and HuCLFOR. Primary PCRs were performed for the VH-CH₁using a mixture of the 6 HuVHBACK2 primers and HulgG1-4 CG1FOR and forthe V lambda-C lambda using a mixture of the 7 HuV BACK primers and HuCFOR. Restriction, cloning and screening proceeded as described. Todetermine the diversity of the clones, the VH and V lambda genes of 15clones were PCR amplified, restricted with the frequent cuttingrestriction enzyme BstN1 and analyzed on a 4% agarose gel (see example20).Assay for anti-Rh-D activity and demonstration of specificity: A 5%(vol/vol) suspension of either Rh-D positive (OR2R2) or Rh-D negative(Orr) erythrocytes in phosphate buffered saline (PBS, pH 7.3) wereincubated with a papain solution for 10 min at 37° C. The erythrocyteswere washed three times in PBS and a 1% (vol/vol) suspension oferythrocytes was made up in PBS supplemented with 1% (vol/vol) of bovineserum albumin (BSA). Fifty ul of a papain treated erythrocyte suspensionand 50 ul of phage supernatant were placed in the wells of round bottommicrotitre plates and the plates were placed on a TItertek plate shakerfor 2 min. After 15 min incubation at 37° C. 100 ul of PBS/BSA was addedto each well. The plates were centrifuged at 200 g for 1 min and thesupernatant was discarded. The erythrocytes were resuspended in theremaining PBS/BSA and the Fab fragments were crosslinked by addition ofthe 9E10 monoclonal antibody (50 ul a 1 ug/ml solution in PBS/BSA)directed against the myc peptide tag (Ward, E. S., et al., Nature 1989.supra). The plates were placed at room temperature (RT) untilsedimentation had occurred. Agglutination of erythrocytes caused adiffuse button of erythrocytes and the results were evaluatedmacroscopically. Specificity was confirmed with a standard prepapainized(as above) panel of 9 erythrocyte suspensions in PBS (all suspensionsblood group O 4 D positive and 5 D negative) known to have homozygousexpression of all the clinically relevant erythrocyte blood groupalloantigens. The number of copies of the D antigen on the D positivecells varied between 10,000 and 20,000 per erythrocyte depending on theRh genotype. Briefly, 50 ul phage supernatant in PBS supplemented with2% (vol/vol) skimmed milk was mixed with 50 ul of a 2% erythrocytesuspension in PBS in glass tubes and incubated for 15 min at 37° C.After one wash with PBS/BSA the erythrocytes were pelleted andresuspended in 50 ul donkey anti-human lambda light chain (Sigma L9527,diluted 1:40 in PBS/BSA). The tubes were centrifuged for 1 min at 200 gand agglutination was read macroscopically using “tip and roll” method.Resultsa PCR assembly of a Fab from a human hybridoma: A single band of thecorrect size was obtained after amplification. Thirty-eight of 96 clones(40%) screened specifically agglutinated Rh-D positive but not Rh-Dnegative red blood cells. The results demonstrate a high frequency ofsuccessful splicing in the assembly process and the potential of thistechnique for one step cloning of human hybridomas.b Assembly of human Fabs from a polyclonal lymphoblastic cell line(LCL): Analysis of the diversity of the clones indicated that 3different heavy chain families and 2 different light chains familieswere present. Five anti-Rh-D specific clones were identified out of 96screened. The VH and VX chains had identical nucleotide sequences ineach clone and were typical of anti-Rh-D V-genes (unpublished results).The results demonstrate the potential of this technique to assemble,clone and isolate human antibody fragments from polyclonal cellpopulations (see also section on isolation of specific bindingactivities from an ‘unimmunized’ human library (examples 42 and 43).

Example 41 Selection of Phage Displaying a Human Fab Fragment DirectedAgainst the Rhesus-D Antigen by Binding to Cells Displaying the Rhesus DAntigen on their Surface

A large number of important antigens are integral components of cellsurface membranes, i.e. they are cell surface antigens. These includetumor specific antigens and red and white blood cell surface antigens.In many instances, it would be important to isolate antibodies againstthese antigens. For example, antibodies directed against the rhesus-D(Rh-D) antigen on red blood cells are used both diagnostically andtherapeutically. Many of these antigens are difficult to purify andsome, like Rh-D, are not biologically active when isolated from themembrane. Thus, it would be useful to be able to affinity purifyantibody fragments displayed on the surface of bacteriophage directly oncell surface antigens. To test the feasibility of affinity purificationon cell surface antigens, the anti-Rh-D human monoclonal antibody Fog-Bwas displayed as a Fab fragment on the surface of bacteriophage fd. Thedisplayed Fog-B Fab fragment bound antigen as determined byagglutination assay and could be affinity purified on the basis of itsbinding on the surface of Rh-D positive red blood cells but not Rh-Dnegative red blood cells.

Materials and Methods

Construction of a clone encoding an anti-Rh-D Fab fragment in phagemidpHENI and display of the Fab fragment on the surface of bacteriophagefd.

The human hybridoma Fog-B has been previously described (N. C.Hughes-Jones et al Biochem, J. 268 135 (1990). It produces anIgG-1/lambda antibody which binds the Rh-D antigen. RNA was preparedfrom 10⁷ hybridoma cells using a modified method of Cathala (asdescribed in example 14) and 1st strand cDNA synthesized using specificimmunoglobulin heavy and light chain primers (HuVH1 FOR [example 40] andHuCλ. FOR (5′-GGA ATT CTT ATG AAG ATT CTG TAG GGG CCA C-3′) (SEQ IDNO:73)) as described in example 14. The VH gene was subsequentlyamplified from an aliquot of the 1st strand cDNA using HuVH4aBACK andHuVH1FOR. The Vλ gene was amplified using a Vλ primer specific for Fog-B(VλFog-B: 5′-AAC CAG CCA TGG CC AGT CTG TGT TGA CGC AGC C-3′) (SEQ IDNO:74). The PCR conditions were as described in example 40. The PCRproducts were analyzed by running 5 μl on a 2% agarose gel. Theremainder was extracted twice with ether, twice with phenol/chloroform,ethanol precipitated and resuspended in 50 μl of H₂O. The amplified VHDNA was digested with Pst1 and BstEII, and the amplified Vλ-Cλ DNA withNco1 and EcoR1. The fragments were purified on a 2% agarose gel,extracted using Geneclean, and sequentially ligated into the solubleexpression vector pJM-1 Fab D1.3 (FIG. 48( i)). Clones containing thecorrect insert were initially identified by restriction analysis andverified by assay of expressed soluble Fab (see example 23 for inductionconditions). The Fog-B Fab cassette was amplified from pJM-1 by PCRusing HuVH4BACK-Sfi and Hu Cλ-Not, digested with the appropriaterestriction enzymes and ligated into pHEN1. Clones containing thecorrect insert were identified initially by restriction analysis andsubsequently by assay (see example 25 for induction conditions).

Assay for soluble Fog-B Fab fragment and phage displayed Fog-B Fabfragment for anti-Rh-D activity and documentation of specificity.

Assay of the soluble expressed Fab was performed on unconcentrated E.coli supernatant. Assay of Fog-B displayed on the phage surface wasperformed on phage that had been concentrated 10 fold by PEGprecipitation and then resuspended in PBS. the assays for activity andspecificity are as described in example.

Cell Surface Affinity Purification of Phage Displaying Fog-B Anti-Rh-DFab Fragment

Purified Fog-B phage was mixed with purified phage Fd-Tet CAT-1displaying the anti-lysozyme scFv D1.3 (pAbD1.3) in a ratio ofapproximately 1 Fog-B:50 scFvD1.3. Prepapainized erythrocytes (OR2R2[Rhesus positive] or Orr [Rhesus negative]) were suspended in PBSsupplemented with 2% skimmed milk powder in a concentration of 4×10⁷/ml.One ml of this suspension was mixed with 10¹¹ phage suspended in 2 ml ofPBS supplemented with 2% skimmed milk and incubated for 30 min at roomtemperature under continuous rotation. The erythrocytes were washedthree times with an excess of ice-cold PBS (10 ml per wash) andsubsequently pelleted. The phage were eluted from the cells byresuspending in 200 μl of 76 mM citric acid pH 2.8 in PBS for 1 min. Thecells were then pelleted by centrifugation for 1 min at 3000 rpm and thesupernatant containing the eluted phage was neutralized by adding 200 μlof 240 mM Tris-base, 22 mM Disodium hydrogen phosphate in 1% w/volalbumin. Serial dilutions of the eluate was used to infect TG1 cells.Fog-B Fab phage were selected on ampicillin plates and scFvD1.3 phage ontetracycline plates and the titre of each determined prior to selection,after selection on rhesus-D negative cells and after selection onrhesus-D positive cells.

Results

Fog-B Fab fragment displayed on the surface of the phage derived fromthe phagemid pHEN clone specifically agglutinated rhesus-D positive butnot rhesus D-negative red blood cells. Affinity purification of theFog-1 Fab phagemid on Rh-D positive red blood cells resulted in anenrichment from 1:50 to 1500:1 (Fog-B Fab:scFvD1.3), whereaspurification on Rh-D negative red blood cells demonstrated essentiallyno enrichment (10 fold).

TITRE Fog-B Fog-B Fab scFvD1.3 FAb/scFvD1.3 RATIO Prior to selection 1.0× 10⁸ 5.0 × 10⁹ 1:50 Selection on Rh-D 2.0 × 10⁴ 1.0 × 10⁵ 1:5  negativecells Selection on Rh-D 6.0 × 10⁶ 4.0 × 10³ 1500:1   positive cells

Example 42 A PCR Based Technique for One Step Cloning of Human scFvConstructs

Assembly of human scFv is similar to the assembly of mouse scFvsdescribed in example 14. To develop the PCR cloning of human V genes itwas necessary to design a new range of human specific oligonucleotideprimers (table 10). The use of these primers for the generation of humanFabs is described in example 40. The assembly of human scFvs isessentially the same but requires a set of FORWARD primers complementaryto the J segments of the VH, VK and V lambda genes. (For Fabs FORWARDprimers complementary to the constant region are used). The J segmentspecific primers were designed based on the published JH, JK and Jlambda sequences (Kabat, E. A. et al, Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987).

In addition, a different linker is needed for scFvs than for Fabs so forhuman scFvs a new set of primers was needed to prepare the linker.Primers complementary to the JH forward primers and the VK and V lambdaback primers were synthesized to permit generation of linker DNA by PCRamplification of plasmid template containing the scFv linker (Table 10,FIG. 49). To ensure adequate amplification, the primers were extendedinto the actual linker sequence. Using these primers to make the scFvlinker DNA, 52 separate PCR reactions were performed using each of the 4reverse JH primers in combination with each of the 13 reverse VK and Vlambda oligonucleotides. The template was approximately ing ofpSW2scD1.3 (Ward, E. S. 1989 supra) containing the short peptide(Gly4Ser)3 (Huston, J. S. et al., Gene 1989. 77:61)

A Specific Example of PCR Assembly of a Human scFv Library

This example describes the generation of a human library of scFvs madefrom an unimmunized human:

500 ml of blood, containing approximately 10⁸ B-cells, was obtained froma healthy volunteer blood donor. The white cells were separated onFicoll and RNA was prepared as described in example 14.

Twenty percent of the RNA, containing the genetic material fromapproximately 2×10⁷ B-cells, was used for cDNA preparation as describedin example 40. Heavy chains originating from IgG and IgM antibodies werekept separate by priming cDNA synthesis with either an IgG specificprimer (HuIgG1-4-CH1FOR) or an IgM specific primer (HuIgMFOR). Aliquotsof the cDNA was used to generate four separate scFv libraries (IgG-K,IgG-lambda, IgM-K and IgM-lambda) as described in example 40. Theresulting libraries were purified on 1.5% agarose, electroeluted andethanol precipitated. For subsequent cloning, the K and lambda librarieswere combined giving separate IgG and IgM libraries.

Cloning of the library: The purified scFv fragments (1-4 ug) weredigested with the restriction enzymes NotI and either Sfil or NcoI.After digestion, the fragments were extracted with phenol/chloroform,ethanol precipitated. The digested fragments were ligated into eitherSfiI-NotI or NcoI-NotI digested, agarose gel electrophoresis purifiedpHEN1 DNA (bug) (see example 24), in a 100 μl ligation mix with 2,000 UT4 DNA ligase (New England Biolabs) overnight at room temperature. Theligation mix was purified by phenol extraction and ethanol precipitated.The ligated DNA was resuspended in 10 μl of water, and 2.5 μl sampleswere electroporated into E. coli TG1 (50 μl). Cells were grown in 1 mlSOC for 1 hr and then plated on 2×TY medium with 100 μg/ml ampicillinand 1% glucose (AMP-GLU), in 243×243 mm dishes (Nunc). After overnightgrowth colonies were scraped off the plates into 10 ml 2×TY containingAMP-GLU and 15% glycerol for storage at −70° C. as a library stock.

Cloning into SfiI-NotI and NcoI-NotI digested pHEN1 yielded libraries of10⁷ and 2×10⁷ clones respectively for the IgM libraries andapproximately 5×10⁷ clones for each of the two IgG libraries.

Example 43 Isolation of Binding Activities from a Library of scFvs froman Unimmunized Human

The ability to select binding activities from human antibody librariesdisplayed on the surface of phage should prove even more important thanisolation of binding activities from murine libraries. This is becausethe standard way of generating antibodies via hybridoma technology hasnot had the success with human antibodies that has been achieved withmouse. While in some instances it will be possible to make librariesfrom immunized humans, in many cases, it will not prove possible toimmunize due to toxicity or lack of availability of an appropriateimmunogen or ethical considerations. Alternatively, binding activitiescould be isolated from libraries made from individuals with diseases inwhich therapeutic antibodies are generated by the immune response.However, in many cases, the antibody producing cells will be located inthe spleen and not available in the circulating pool of peripheral bloodlymphocytes (the most easily accessible material for generating thelibrary). In addition, in diseases associated with immunosuppression,therapeutic antibodies may not be produced.

An alternative approach would be to isolate binding activities from alibrary made from an unimmunized individual. This approach is based onestimates that a primary repertoire of 10⁷ different antibodies islikely to recognize over 99% of epitopes with an affinity constant of10⁵ M⁻¹ or better. (Pewrelson, A. S. Immunol. Rev, (1989) 110:5). Whilethis may not produce high affinity antibodies, affinity could be boostedby mutation of the V-genes and/or by using the isolated VH domain in ahierarchical approach with a library of light chains (or vice versa). Inthis section, we demonstrate the feasibility of this approach byisolating specific antigen binding activities against three differentantigens from a library of scFvs from an unimmunized human.

Materials and Methods

The generation of the human scFv library used for the isolation ofbinding activities described in this example is detailed in example 42.

Estimation of diversity of original and selected libraries: Recombinantclones were screened before and after selection by PCR (example 20) withprimers LMB3 (which sits 5′ of the pelB leader sequence and is identicalto the reverse sequencing primer (−40 n) of pUC19) and fd-SEQ1 (seeexample 37) followed by digestion with the frequent-cutting enzymeBstN1. Analysis of 48 clones from each unselected library indicated that90% of the clones had inset, and the libraries appeared to be extremelydiverse as judged by the BstNI restriction pattern.Rescue of Phagemid libraries for enrichment experiments: To rescuephagemid particles from the library, 100 ml 2×TY containing AMP-GLU (seeexample 42) was inoculated with 10⁹ bacteria taken from the library(prepared in example 42) (approx. 10 μl) and grown for 1.5 hr, shakingat 37° C. Cells were spun down (IEC− centrifuge, 4 K, 15 min) andresuspended in 100 ml prewarmed (37° C.) 2×TY− AMP (see example 41)medium, 2×10¹⁰ pfu of VCS-M13 (Stratagene) particles added and incubated30 min at 37° without shaking. Cells were then transferred to 900 ml2×TY containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml)(AMP-KAN), and grown overnight, while shaking at 37° C. Phage particleswere purified and concentrated by three PEG-precipitations (seematerials and methods) and resuspended in PBS to 10¹³ TU/ml (ampicillinresistant clones).Enrichment for phOx:BSA binders by selection on tubes: For enrichment, a75×12 mm Nunc-immunotube (Maxisorp; Cat. No. 4-44202) was coated with 4ml phOx:BSA (1 mg/ml; 14 phOx per BSA in 50 mM NaHCO₃ pH 9.6 buffer)overnight at room temperature. After washing three times with PBS, thetube was incubated for 2 hr at 37° C. with PBS containing 2% Marvel (2%MPBS) for blocking. Following three PBS washes, phagemid particles (10¹³TU) in 4 ml of 2% MPBS were added, incubated 30 min at room temperatureon a rotating turntable and left for a further 1.5 hours. Tubes werethen washed with 20 washes of PBS, 0.1% Tween 20 and 20 washes PBS (eachwashing step was performed by pouring buffer in and out immediately).Bound phage particles were eluted from the tube by adding 1 ml 100 mMtriethylamine pH 11.5 and rotating for 15 min. The eluted material wasimmediately neutralised by adding 0.5 ml 1.0 M Tris-HCl, pH 7.4 andvortexed. Phage was stored at 4° C.

Eluted phage (in 1.5 ml) was used to infect 8 ml logarithmic growing E.coli TG1 cells in 15-ml 2×TY medium, and plated on AMP-GLU plates asabove yielding on average 10⁷ phage infected colonies.

For selection of phOx:BSA binders, the rescue-tube enrichment-platingcycle was repeated 4 times, after which phagemid clones were analysedfor binding by ELISA.

Enrichment for lysozyme binders by panning and on columns: A petri dish(35×10 mm Falcon 3001 Tissue culture dish) was used for enrichment bypanning. During all steps, the plates were rocked on an A600 rockingplate (Raven Scientific). Plates were coated overnight with 1 ml turkeyegg white lysozyme (3 mg/ml) in 50 mM sodium hydrogen carbonate (pH9.6), washed three times with 2 ml PBS, and blocked with 2 ml 2% MPBS atroom temperature for 2 hours. After three PBS washes approximately 10¹²TU phage particles in 1 ml 2% MPBS were added per plate, and leftrocking for 2 hr at room temperature. Plates were washed for 5 min with2 ml of the following solutions: 5 times PBS, PBS-Tween (0.02%Tween-20), 50 mM Tris-HCl (pH 7.5)+500 mM NaCl, 50 mM Tris-HCl (pH8.5)+500 mM NaCl, 500 mM Tris-HCl (pH 9.5)+500 mM NaCl and finally 50 mMsodium hydrogen carbonate pH 9.6 Bound phage particles were then elutedby adding 1 ml 100 mM triethylamine pH 11.5 and rocking for 5 min beforeneutralising with 1 M Tris-HCl (pH 7.4) (as above). Alternatively, 1 mlturkey egg white lysozyme-Sepharose columns were used for affinitypurification (McCafferty, J., et al., Nature 1990. 348: 552) Columnswere washed extensively with PBS, blocked with 15 ml 2% MPBS, and phage(10¹² TU) in 1 ml 2% MPBS loaded. After washing with 50 ml PBS, 10 mlPBS− Tween (PBS+0.02% Tween-20), 5 ml of 50 mM Tris-HCl (pH 7.5)+500 mMNaCl, 5 mM Tris-HCl 9pH 8.5)+500 mM NaCl, 5 ml of 50 mM Tris-HCl (pH9.5)+500 mM NaCl and finally 5 ml of 50 mM sodium hydrogen carbonate pH9.6. Bound phage was eluted using 1.5 ml 100 mM triethylamine andneutralised with 1 M Tris-HCl (pH 7.4).

For selection of turkey egg white lysozyme binders, the rescue-tubeenrichment-plating cycle or rescue-column-plating cycle was repeated 4times, after which phagemid clones were analysed for binding by ELISA.

Rescue of individual phagemid clones for ELISA: Clones resulting fromreinfected and plated phage particles eluted after 4 rounds ofenrichment, were inoculated into 150 μl of 2×TY-AMP-GLU in 96-wellplates (cell wells, Nunclon), grown with shaking (250 rpm) overnight at37° C. A 96-well plate replicator (‘plunger’) was used to inoculateapproximately 4 μl of the overnight cultures on the master plate into200 μl fresh 2×TY-AMP-GLU. After 1 hr, 50 μl 2×TY-AMP-GLU containing 10⁸pfu of VCS-M13 was added to each well, and the plate incubated at 37° C.for 45 min, followed by shaking the plate at 37° C. for 1 hr. Glucosewas then removed by spinning down the cells (4K, 15 min), and aspiratingthe supernatant with a drawn out glass pasteur pipette. Cells wereresuspended in 200 μl 2×TY-AMP-KAN (Kanamycin 50 ug/ml) and grown 20 hr,shaking 37° C. Unconcentrated supernatant containing phage was taken foranalysis by ELISA.ELISAAnalysis for binding to phOx:BSA, BSA or lysozyme was performed by ELISA(see example 9), with 100 μg/ml phOx:BSA or BSA, or 3 mg/ml turkey eggwhite lysozyme used for coating. Determination of cross reactivity tounrelated antigens with the isolated clones was also determined by ELISAon plates coated with 100 ug/ml of an irrelevant antigen (keyhole limpethaemocyanin (KLH), ovalbumin, chymotrypsinogen, cytochrome C,thyroglobulin, GAP-DH (glyceraldehyde-3-phosphate dehydrogenase), ortrypsin inhibitor).Characterization of ELISA positive clones: All antigen specific clonesisolated were checked for cross reactivity against a panel of irrelevantantigens as described above. The diversity of the clones was determinedby PCR screening as described above and at least two clones from eachrestriction pattern were sequenced by the dideoxy chain terminationmethod.ResultsIsolation and characterization of phOx:BSA binders: After 4 rounds ofselection, ELISA-positive clones were isolated for phOx:BSA. All clonesoriginated from the IgM library. Of 96 clones analysed, 43 clones werebinding to both phOx:BSA and BSA, with ODs ranging from 0.4 to 1.3(background 0.125). These clones are designated as BSA binders. Thebinding to BSA seemed to be specific, since none of the 11 clonesanalysed gave a signal above background when used in an ELISA with KLH,ovalbumin, chymotrypsinogen, cytochrome C, lysozyme, thyroglobulin,GAP-DH, or trypsin inhibitor. all BSA binding clones had the same BstNIrestriction pattern, and 14 clones were completely sequenced. Thirteenof the fourteen clones had the same sequence, the VH was derived from ahuman VH3 family gene and the VL from a human V lambda 3 family gene(Table 1). The other BSA binder was derived from a human VH4 family geneand a human Vk1 family gene (data not shown).

One clone was isolated which bound to phOx:BSA only (OD 0.3), and boundphage could be completed off completely by adding 0.02 mM4-ε-amino-caproic acid methylene 2-phenyl-oxazol-5-one (phOx-CAP) as acompetitor. Also no binding above background could be detected to thepanel of irrelevant proteins described above. The sequence revealed a VHderived from a human VH1 family gene and a VL derived from a human Vlambda 1 family gene (Table 11).

Isolation and characterisation of lysozyme binders: After 4 rounds ofselection, 50 ELISA-positive clones were isolated for turkey lysozyme.The majority of the clones, greater than 95%, were from the IgM library.The binding to lysozyme seemed to be specific, since none of the clonesanalysed gave a signal above background when used in an ELISA with KLH,ovalbumin, chymotrypsinogen, cytochrome C, thyroglobulin, GAP-DH, ortrypsin inhibitor. The lysozyme binding clones gave 3 different BstNIrestriction patterns, and at least 2 clones from each restrictionpattern were completely sequenced. The sequences indicated the presenceof 4 unique human VH-VL combinations. (Table 11).Conclusion

The results indicate that antigen binding activities can be isolatedfrom repertoires of scFvs prepared from IgM cDNA from human volunteersthat have not been specifically immunized.

Example 44 Rescue of Human IgM Library Using Helper Phage Lacking Gene 3(δg3)

This example describes the rescue of gene 3 fusions from a human libraryusing a helper phage with a gene 3 deletion.

100 μl of bacterial stock of the IgM phagemid library prepared asdescribed (example 42), containing 5×10⁸ bacteria, was used to inoculate100 mls of 2×TY medium containing 100 μg/ml ampicillin, 2% glucose(TY/Amp/Glu). This was grown at 37° C. for 2.5 hours. 10 mls of thisculture was added to 90 mls of prewarmed TY/Amp/Glu and infectioncarried out by adding 10 mls of a 200 fold concentrate of KO7 helperphage lacking gene 3 (M13KO7gIIIΔ No. 3) (example 34) and incubating for1 hour at 37° C. without shaking. Preparation of M13KO7gIII No. 3 was asdescribed in example 34. After centrifugation at 4,000 r.p.m. for 10minutes the bacteria were resuspended in 100 mls of 2×TY mediumcontaining 100 μg/ml ampicillin (with no glucose). Titration of theculture at this point revealed that there were 1.9×10⁸ infected bacteriaas judged by their ability to grow on plates containing both ampicillin(100 μg/ml) and kanamycin (50 μg/ml). Incubation was continued for 1hour with shaking before transferring to 2.5 liters of 2×TY mediumcontaining 100/μg/ml ampicillin, 50 μg/ml kanamycin, contained in five2.5 liter flasks. This culture was incubated for 16 hours and thesupernatant prepared by centrifugation. (10-15 minutes at 10,000 r.p.m.in a Sorvall RC5B centrifuge at 4° C.). Phage particles were harvestedby adding ⅕th volume of 20% polyethylene glycol, 2.5 M NaCl, standing at4° C. for 30 minutes and centrifuging as above. The resulting pellet wasresuspended in 40 mls of 10 mM Tris, 0.1 mM EDTA pH 7.4 and bacterialdebris removed by centrifugation as above. The packaged phagemidpreparation was then re-precipitated, collected as above and resuspendedin 10 mls of 10 mM Tris, 0.1 mM EDTA pH 7.4. The liter of thispreparation was 4.1×10″ transducing units/ml (ampicillin resistance).

Tubes coated with OX-BSA were prepared as described in example 45 forpanning the phagemid library from example 42. The rescued library wasalso panned against tubes coated with bovine thyroglobulin (Sigma).These were coated at a concentration of 1 mg/ml thyroglobulin in 50 mMNaHCO3 pH9.6 at 37° C., overnight. Tubes were blocked with PBScontaining 2% milk powder (PBS/M) and incubated with 1 ml of the rescuedphagemid library (the equivalent of 250 mls of culture supernatant)mixed with 3 mls of PBS/M for 3 hours. Washing, elution, neutralisationand infection were as described in example 45.

Results: Panning Against Oxazalone—BSA

The first round of panning against OX-BSA yielded 2.8×10⁶ phage. A largebacterial plate with 1.4×10⁶ colonies derived from this eluate wasscraped into 10 mls of 2×TY, 20% glycerol, shaken for 10 minutes,aliquoted and stored. This was also used to inoculate a fresh culturefor rescue with M13KO7gIII No. 3. (Bacteria and rescued phage derivedfrom first round panning against OX-BSA are named OXPAN1. Bacteria orrescued phage derived from second and third round pannings are namedOXPAN2 and OXPAN3 respectively) Rescue of phagemid with M13KO7gIII No. 3after each round of panning was essentially as described above but using5 ml volumes for the initial cultures in TY/Amp/Glu, using 1 ml ofhelper phage and transferring to 100-500 mls of 2×TY medium containing100 μg/ml ampicillin, 50 μg/ml kanamycin. Second and third round panningsteps were as described above for the first round, but using 0.8-1.0 mlsof 100 fold concentrated phage (the equivalent of 80-100 mls of culturesupernatant). The eluate from the second round panning contained 8×10⁸infectious particles and the eluate from the third round panningcontained 3.3×10⁹ infectious particles.

Panning Against Thyroglobulin

The first round panning against thyroglobulin yielded 2.52×10⁵infectious particles. Half of the eluate was used to generate 1.26×10⁵bacterial colonies on a large plate. These colonies were scraped into 10mls of 2×TY, 20% glycerol, shaken for 10 minutes, aliquoted and stored.These bacteria and rescued phage derived from them are termed THYPAN1,and used to inoculate a fresh culture for rescue with M13KO7gIII No. 3to give a polyclonal rescued phage preparation. Material similarlyderived from second and third round pannings are termed THYPAN2 andTHYPAN3 respectively. Second and their round pannings with thyroglobulinwere as described for second and third round OX-BSA panning. The eluatefrom the second round panning contained 8×10⁷ transducing units and theeluate from the third round panning contained 6×10⁷ infectiousparticles.

Elisa Screening of Clones Derived by Panning

40 colonies derived form the third round of panning againstthyroglobulin (THYPAN3) were picked into a 96 well plate and grownovernight at 37° C. in 200 μl of TY/Amp/Glu. Similarly 48 colonies fromtwo rounds and 48 colonies from three rounds of panning against OX-BSAwere grown (OX-PAN2 and OX-PAN3). Polyclonal phage were prepared at thesame time. Next day 5 μl from each culture was transferred to 100 μl offresh prewarmed TY/Amp/Glu grown for 1.5 hours and M13KO7gIII No. 3added (2×10⁵ infectious phage per well in 100 μl of TY/Amp/Glu). thesewere incubated for 1 hour at 37° C. without shaking, centrifuged at4,000 r.p.m. for 10 minutes, resuspended in 150 μl of 2×TY mediumcontaining 100 μg/ml ampicillin and incubated for a further hour withshaking before adding to 2 mls of medium containing 100 μg/mlampicillin, 50 μg/ml kanamycin. After overnight growth the cultures werecentrifuged at 4,000 r.p.m. for 10 minutes and the supernatantscollected. ELISA plates used to screen THYPAN3 clones were coated at 37°C. overnight with 200 μg/ml thyroglobulin in 50 mM NaHCO₃pH9.6. Platesused for OXPAN2 and OXPAN3 were coated at 100 μg/ml OX-BSA in PBS at 37°C. overnight.

120 μl of culture supernatant was mixed with 30 μl of 5×PBS, 10% milkpowder and incubated at room temperature for 2 hours at roomtemperature. ELISAs were carried out as described in example 18.

For thyroglobulin, 18 out of 40 clones were positive (0.3-2.0 O.D. after30 minutes). (A phage control (vector pCAT3) gave a reading of 0.07O.D.). In addition, positives were also seen on the polyclonal phagepreparations THYPAN1 (0.314 O.D.) and THYPAN2 (0.189 O.D.) compared withphage derived from the original non-panned phagemid library (0.069O.D.). All polyclonal phage were PEG precipitated and used at a 10 foldconcentration.

PCR reactions and BstN1 digests were carried out on the positive clonesas described above and six different patterns of DNA fragments wereobtained showing that at least six different clones had been isolated.

For OX-BSA after two rounds of panning, 30 of 48 clones were positive byELISA and after three rounds, 42 of 48 were positive. In a separateexperiment, positive signal was obtained from the polyclonal phagepreparations OXPAN1 (0.9880D) and OXPAN2 (1.7170D) compared with phagederived from the original non-panned phagemid library (0.186 O.D.) after30 minutes.

Specificity of Clones for Thyroglobulin or OX-BSA

Selected clones (11 anti-thyroglobulin, 5 anti-OX-BSA) representing eachof the different BstNI restriction digest patterns were assayed forbinding to a panel of irrelevant antigens. ELISA plates were coated withantigen (100 μl/ml in 50 mM NaHCO₃, pH 9.6) by overnight incubation at37° C. The panel of antigens consisted of keyhole limpet haemocyanin,hen egg lysozyme, bovine serum albumin, ovalbumin, cytochrome c,chymotrysinogen, trypsin inhibitor, GAP-D11 (glyceraldehyde-3-phosphatedehydrogenase), bovine thyroglobulin and oxazolone-BSA. Duplicatesamples of phage supernatant (80 μl+20 μl 5×PBS, 10% milk powder) wereadded to each antigen and incubated for 1 hour at room temperature. theELISA was carried out as described in example 18.

Each of the thyroglobulin specific clones (11 from 11) were positive forthyroglobulin (OD 0.12-0.76) but after 60 minutes showed no binding(OD<0.03) to any of the 9 irrelevant antigens. Similarly of the 5 Ox-BSAspecific clones 3 had an OD 0.07-0.52 compared to ODs<0.02 for theirrelevant antigens. None of the 5 clones had any binding to BSA alone.

Thus positive clones can be isolated after only two rounds of panning byrescuing with M13K07gIII No. 3. In addition there is a greaterlikelihood with this helper of generating phage particles with more thanone intact antibody molecule. This will potentially increase the avidityof phage-antibodies and may enable isolation of clones of weakeraffinity.

Example 45 Alteration of Fine Specificity of scFv D1.3 Displayed onPhage by Mutagenesis and Selection on Immobilised Turkey Lysozyme

The D1.3 antibody binds hen egg lysozyme (HEL) with an affinity constantof 4.5×10⁷M⁻¹ whereas it binds turkey egg lysozyme (TEL) with anaffinity of <1×10⁵M⁻¹. (Harper et al (1987) Molecular Immunology 24 p97-108, Amit et al (1986) Science 233 p 747-753).

It has been suggested that this is because the glutamine residue presentat position 121 of HEL (gln121) is represented by histidine residue atthe same position in TEL. Thus mutagenising the D1.3 antibody residueswhich interact with gln121 of HEL may facilitate binding to TEL.

According to Amit et al, supra, tyrosine at amino acid position 32,phenylalanine at position 91 and tryptophan at position 92 of the lightchain interact with gln121 of HEL. In addition tyrosine at position 101of the heavy chain also interacts. None of these residues are predictedto be involved in determining the main chain conformation of theantibody variable regions (Chothia and Lesk (1987) Journal of MolecularBiology 196, p 901-917).

Mutagenesis of pCAT3SCFvD1.3

The oligonucleotides mutL91,92, was prepared to randomise phenylalanineat position 91 (L91) and tryptophan at position 92 (L92) of the lightchain. The oligonucleotides mutL32, was prepared to randomise tyrosineat light chain position 32 (L32) and the oligonucleotides mutH101 wasprepared to randomise tyrosine at position 101 of the heavy chain(H101). mutL91,92:

5′ CGT CCG AGG AGT ACT NNN NNN ATG TTG ACA GTA ATA 3′ (SEQ ID NO:75)

mutL32:

5′ CTG ATA CCA TGC TAA NNN ATT GTG ATT ATT CCC 3′ (SEQ ID NO:76)

mutes 101:

5′ CCA GTA GTC AAG CCT NNN ATC TCT CTC TCT GGC 3′(SEQ ID NO:77)

(N represents a random insertion of equal amounts of A, C, G or T) invitro mutagenesis of the phagemid vector, pCAT3scFvD1.3 (example 17)with the oligonucleotide mutL91,92 was carried out using an in vitromutagenesis kit (Amersham). The resultant DNA was transformed byelectroporation into TG1 cells using a Bio-Rad electroportor. 78,000clones were obtained and these were scraped into 15 mls of 2×TY/20%glycerol. This pool was called D1.3L91L92. Single stranded DNA wasprepared by rescue with M13K07 as described in Sambrook et al, 1989supra, and sequenced with the primer FDTSEQ1, using a Sequenasesequencing kit (United States Biochemical Corporation).

This revealed that the DNA had been successfully mutagenised as judgedby the presence of bands in all four DNA sequencing tracks at thenucleotide positions encoding L91 and L92. This mutagenised singlestranded DNA was subjected to a further round of mutagenesis as aboveusing either mutL32 or mutH101 oligonucleotides. Mutagenesis with mutL32gave rise to 71,000 clones (pool called D1.3L32) while mutH101 gave102,000 clones (pool called D1.3H101). These clones were scraped into 15mls of 2×TY/20% glycerol. Single stranded DNA derived from each pool wassequenced with the oligonucleotides D1.3L40 and LINKSEQ1 respectively,as described above, and shown to be correctly randomised.

D1.3L40:

5′ CAG GAG CTG AGG AGA TTT TCC 3′

LINKSEQ1:

5′ TCC GCC TGA ACC GCC TCC ACC 3′

Preparation of Rescued Phage for Affinity Purification

10-20 μl of bacteria derived from each mutagenised pool (plate scrapes)was used to inoculate 5 mls of TY/Glu/Amp. All bacterial growth was at37° C. After 2-3 hours growth, 1 ml was diluted in 5 mls of prewarmedTY/Glu/Amp and infected by addition of 0.5 mls of a 200 fold concentrateof the M13K07gIII Δ No. 3 preparation described in example 34. After 1hour of infection the cultures were centrifuged at 4,000 r.p.m. for 10minutes, resuspended in 2×TY, 100 μg/ml ampicillin, incubated for afurther hour, transferred to 500 mls of 2×TY medium containing 100 μg/mlampicillin, 50 μg/ml kanamycin and grown for 16 hours. The remainingsteps of phage preparation were as described in example 44. Phage werefinally dissolved in 10 mM Tris, 1 mM EDTA pH7.4 at 1/100th the originalculture volume.

Affinity Purification

10 mls of turkey egg lysozyme at a concentration of 10 mg/ml in 0.1MNaHCO3, 0.5MNaCl pH8.3 was mixed with an equal volume of swollenCyanogen Bromide Activated Sepharose 4B (Pharmacia), covalently linkedand washed according to manufacturers instructions. Before use thismatrix (TEL-Sepharose) was washed with 100 volumes of PBS followed by 10volumes of PBSM. The TEL-Sepharose was resuspended in an equal volume ofPBSM and 1 ml was added to 1 ml of a 50 fold concentrate of phage inPBSM and incubated on a rotating platform for 30 minutes at roomtemperature. The actual phage used for this step was prepared by mixingequal volumes of the independent preparations of the three randomisedpools (D1.3L9192, D1.3H101 and D1.3L32). After this binding step, thesuspensions were loaded onto a disposable polypropylene column(Poly-Prep columns, Bio-Rad) and washed with 200 volumes of PBScontaining 0.1% Tween 20. Bound phage were eluted with 1 ml of 100 mMtriethylamine and neutralised with 0.5 ml μM Tris (pH7.4). A dilutionseries was prepared from the eluate and used to infect TG1 cells andplated out on TY plates containing 100 μg/ml ampicillin, 2% glucose.Plates carrying approximately 10⁶ colonies were scraped into 3 mls of2×TY, 20% glycerol and stored at −70° C. 10 μl of this was used toinitiate a second round culture which was rescued with M13K07gIIIΔ No. 3as described above (using a final culture volume of 100 mls). Second andthird round affinity column purification steps were carried out asdescribed above for the first round.

Analysis by ELISA

40 colonies derived from the third round of column purification onTEL-Sepharose were picked into a 96 well plate and grown overnight at37° C. in 200 μl of TY/Amp/Glu. Phagemid particles were rescued andprepared for ELISA as described in example 18. ELISA plates were coatedovernight at 37° C. with hen egg lysozyme (HEL) or turkey egg lysozyme(TEL) at a concentration of 200 μg/ml in 50 mM NaHCO₃ pH9.6 ELISAs werecarried out as described in example 18.

After 15 minutes incubation in substrate, 13 clones were found to benegative (OD<0.05 on HEL and TEL). In all positives, a signal of0.1-0.78 was scored on HEL with the exception of one where signal on HELwas 0.078 but signal on TEL (OD 0.169) brought it in to the positivegroup. The control phagemid preparation had a percentage ratio of signalTEL:HEL of 22%. Clones were deemed to have an unaltered binding if theratio of TEL:HEL was less than 40%. 9 clones fell into this category. 18samples were scored as having altered binding with a ratio of signal onTEL:HEL of between 40-200%.

A dilution series was made on 10 clones which were analysed by ELISA in6 of these clones the profile of binding to HEL was the same as theoriginal clone (pCAT3SCFvD1.3) while the signal with TEL was increased(see FIG. 50( i) clone B1). In the remaining 4 clones, the increasedsignal with TEL was accompanied by a decrease in signal on HEL (see FIG.50 clone A4).

Competition with Soluble Antigen

All of the isolated clones retained binding to HEL to varying extents.In order to determine whether a soluble antigen could compete with theimmobilised antigen, a parallel experiment was carried out, as above,but with the addition of hen egg lysozyme (1 mg/ml) to TEL-Sepharosebefore incubating with the phage preparation. This experiment wascarried through 3 rounds of column purification and 40 colonies werepicked. None of these clones bound HEL or GEL demonstrating that thesoluble antigen had been successful in competing out binding to theimmobilised antigen.

Example 46 Modification of the Specificity of an Antibody by Replacementof the VLK Domain by a VLK Library Derived from an Unimmunised Mouse

When an antibody specificity is isolated it will often be desirable toalter some of its properties particularly its affinity or specificity.This example demonstrates that the specificity of an antibody can bealtered by use of a different VL domain derived form a repertoire ofsuch domains. This method using display on phage would be applicable toimprovement of existing monoclonal antibodies as well as antibodyspecificities derived using phage antibodies. This example shows thatreplacement of the VL domain of scFvD1.3 specific for Hen eggwhitelysozyme (HEL) with a library of VL domains allows selection of scFvfragments with bind also to Turkey eggwhite lysozyme (TEL). Moregenerally this experimental approach shows that specificities ofantibodies can be modified by replacement of a variable domain and givesa further example of the hierarchical approach to isolating antibodyspecificities.

The D1.3 heavy chain was amplified from an existing construct(pSW1-VHD1.3, Ward et al., 1989 supra) by PCR using the primers VH1BACKand VH1FOR, the light chain library was amplified from a cDNA libraryderived from the spleen of an unimmunised mouse, which was synthesizedby using the MJKFONX primers 1, 2, 4, 5 for the first strand as inexample 14. The subsequent amplification was performed with the sameforward primers and the VK2BACK primer. The PCR assembly of the D1.3heavy chain with the light chain library was mediated by the signalchain Fv linker as described in example 14.

Cloning the assembled PCR products (scFv sequences) was done after anadditional PCR step (pull-through) using a BACK primer providing anApaLI site and forward primers which contained a Not 1 site as describedin example 14. ApaL1/Not 1 digested PCR fragments were cloned into thesimilarly digested vector fdCAT2 as in example 11. 5×10⁵ transformationswere obtained after electroporation of the ligation reaction into MC1061cells.

Screening of the phage library for TEL binders was performed by panning.Polystyrene Falcon 2058 tubes were coated (16 hrs) with 2 ml of TEL-PBS(3 mg/ml) and blocked for 2 hrs with 4 ml MPBS (PBS containing 2%skimmed milk powder). Phage derived from the library (5×10¹⁰ transducingunites) in 2 ml of MPBS (2%) were incubated in these tubes for 2 hrs atroom temperature. The tubes were washed 3× with PBS, 1× with 50 mMTris-HCl, pH 7.5, 0.5 M NaCl; 1× with 50 mM Tris-HCl, pH8.5, 0.5 M NaCl,50 mM Tris-HCl, pH 9.5 M NaCl. Finally phage were eluted with 100 mMtriethylamine. Eluted phages were taken to infect TG1 cells, the cellswere plated on 2×TY plates containing 15 μg/ml tetracycline and grownfor 16 h. The colonies were scraped into 25 ml of 2×TY medium and thephages were recovered by PEG precipitation. After a second round ofselection for TEL binders ELISAs were performed as described (example2).

Analysis of 100 clones from the library before affinity selection byELISA on plates coated with TEL showed no binders. In contrast, aftertwo rounds of selection for TEL binding phages about 10% of the phageclones showed positive ELISA signals. ELISA signals were scored positivewith values at least two fold higher than the fdCAT2 vector withoutinsert. A more detailed analysis of binding properties of TEL bindingphages is shown in FIG. 51.

As shown in FIG. 51, several clones were found which bind equally to TELand HEL in contrast to the original D1.3 scFv, which binds almostexclusively to HEL. None of the clones bound to BSA. These findingsindicate that the specificity of these scFvs was broader in comparisonto D1.3, since both lysozymes (HEL and TEL) are recognized, butspecificity for lysozyme was retained since other BSA was notrecognized. The deduced amino acid sequences (derived by DNA sequencing)of two light chains from clones MF1 and M21, which correspond to clones3 and 9 in FIG. 51 are shown in FIG. 52.

In the case of isolated antibodies the experimental approach asdescribed in this study may be particularly useful if recognition of awider range of different but closely related antigens is desired. Forexample, monoclonal antibodies against viral antigens viral antigenslike V3 loop of HIV-1 gp120 are in most cases quite specific for oneparticular virus isolate because of the variability in this part of theHIV-1 env gene. The modification of such antibodies in the way describedin this example may lead to antibodies which cross react with a widerrange of HIV-1 isolates, and would therefore be of potentially highertherapeutic or diagnostic value.

A similar approach could be taken in which a light chain variable domainof desired properties is kept fixed and combined with a library of heavychain variable domains. Some heavy chains, for example VHD1.3 retainbinding activity as single domains. This may allow a strategy where VHdomains are screened for binding activity when expressed on phage andthen binding domains combined with a library of VL domains for selectionof suitable light chain partners.

Example 47 Selection of a Phage Antibody Specificity by Binding to anAntigen Attached to Magnetic Beads. Use of a Cleavable Reagent to AllowElution of Bound Phage Under Mild Conditions

When a phage antibody binds to its antigen with high affinity or avidityit may not be possible to elute the phage antibody from an affinitymatrix with a molecule related to the antigen. Alternatively, there maybe no suitable specific eluting molecule that can be prepared insufficiently high concentration. In these cases it is necessary to usean elution method which is not specific to the antigen-antibody complex.Unfortunately, some of the non-specific elution methods disrupt phagestructure, for instance phage viability is reduced with time at pH12(Rossomando, E. F. and Zinder, N. D. J. Mol. Biol. 36 387-399 1968). Amethod was therefore devised which allows elution of bound phageantibodies under mild conditions (reduction of a dithiol group withdithiothreitol) which do not disrupt phage structure.

Target antigen was biotinylated using a cleavable biotinylation reagent,BSA conjugated with 2-phenyl-5-oxazolone (0. Makela et al. supra) wasmodified using a biotinylation reagent with a cleavable dithiol group(sulphosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate fromPierce) according to the manufacturers instructions. This biotinylatedantigen was bound to streptavidin coated magnetic beads and the complexused to bind phage. Streptavidin coated magnetic beads (Dynal) wereprecoated with antigen by mixing 650 μg of biotinylated OX-BSA in 1 mlPBS, with 200 μl of beads for at least 1 hour at room temperature. Freeantigen was removed by washing in PBS. One fortieth of the complex(equivalent to 5 μl of beads and an input of 17.5 μg of OX-BSA) wasadded to 0.5 ml of phage in PBSM (PBS containing 2% skimmed milk powder)containing 1.9×10¹⁰ phage particles mixed at the ratios of pAbD1.3directed against lysozyme (example 2) to pAbNQ11 directed against2-phenyl-5-oxazolone (example 11) shown in Table 12.

After 1 hour of incubation with mixing at room temperature, magneticbeads were recovered using a Dynal MPC-E magnetic desperation device.They were then washed in PBS containing 0.5% Tween 20, (3×10 minutes,2×1 hour, 2×10 minutes) and phage eluted by 5 minutes incubation in 500PBS containing 10 mM dithiothreitol. The eluate was used to infect TG 1cells and the resulting colonies probed with the oligo NQ11CDR3

(5′ AAACCAGGCCCCGTAATCATAGCC 3′) (SEQ ID NO:80)

derived from CDR3 of the NQ11 antibody (This hybridises to pAbNO11 butnot pAb D1.3).

A 670 fold enrichment of pAbNQ11 (table 12) was achieved form abackground of pAbD1.3 in a single round of purification using theequivalent of 17.5 μg of biotinylated OX-BSA.

This elution procedure is just one example of an elution procedure undermild conditions. A particularly advantageous method would be tointroduce a nucleotide sequence encoding amino acids constituting arecognition site for cleavage by a highly specific protease between theforeign gene inserted, in this instance a gene for an antibody fragment,and the sequence of the remainder of gene III. Examples of such highlyspecific proteases are Factor X and thrombin. After binding of the phageto an affinity matrix and elution to remove non-specific binding phageand weak binding phage, the strongly bound phage would be removed bywashing the column with protease under conditions suitable for digestionat the cleavage site. This would cleave the antibody fragment from thephage particle eluting the phage. These phage would be expected to beinfective since the only protease site should be the one specificallyintroduced. Strongly binding phage could then be recovered by infectinge.g. E. coli TG1 cells.

Example 48 Use of Cell Selection to Provide an Enriched Pool of AntigenSpecific Antibody Genes. Application to Reducing the Complexity ofRepertoires of Antibody Fragment Displayed on the Surface ofBacteriophage

There are approximately 10¹⁴ different combinations of heavy and lightchains derived from the spleen of an immunised mouse. If the randomcombinatorial approach is used to clone heavy and light chain fragmentsinto a single vector to display scFv, Fv or Fab fragments on phage, itis not a practical proposition to display all 10¹⁴ combinations. Oneapproach, described in this example, to reducing the complexity is toclone genes only from antigen selected cells. (An alternative approach,which copes with the complexity is the dual combinatorial librarydescribed in example 26).

The immune system uses the binding of antigen by surface immunoglobulinto select the population of cells that respond to produce specificantibody. This approach of selecting antigen binding cells has beeninvestigated to reduce the number of combinatorial possibilities and soincrease the chance of recovering the original combination of heavy andlight chains.

The immunological response to the hapten 4-hydroxy-3-nitrophenylaceticacid (NP) has been extensively studied. Since the primary immuneresponse to NP uses only a single light chain the applicants were ableto examine the use of the combinatorial method using a fixed light chainand a library of heavy chains to examine the frequencies genes that codefor antibodies binding to NIP (4-hydroxy-3-iodo-5-nitrophenylaceticacid). The applicants have thus used this system to investigate themerits of selecting cell populations prior to making combinatoriallibraries for display on phage.

Methods

2.1 Hapten Conjugates

Chick gamma globulin (CGG, Sigma, Poole, UK) and Bovine serum albumen(BSA, Boehringer, Mannheim, Germany) were conjugated withNP-O-succinimide or NIP-caproate-O-succinimide (Cambridge ResearchBiochemicals, Northwich, UK) based on the method described by Brownstone(Brownitone, A., Mitchison, N. A. and Pitt-Rivers, R., Immunology 1966.10: 465-492). The activated compounds were dissolved indimethylformamide and added to proteins in 0.2 M sodium hydrogencarbonate. They were mixed with constant agitation for 16 hours at 4° C.and then dialysed against several changes of 0.2 M sodium hydrogencarbonate. They were finally dialysed into phosphate buffered saline(PBS). The conjugates made were NP₁₂CGG, NIP₁₀BSA. The NIP₁₀BSAderivative was subsequently biotinylated using a biotinylation kitpurchased from Amersham (Amersham International, Amersham, UK).

2.2 Animals and Immunisation

Mice of the strain C57BL/6 were immunised by intraperitoneal injectionof 100 μg NP-CGG in Complete Freunds Adjuvant at 10 weeks of age.

2.3 Spleen Preparation

Seven days after immunization cells from the spleen were prepared asdescribed by Galfre and Milstein (Galfre, G. and Milstein, C. MethodsEnzymol. 1981. 73:3-46). Red cells were lysed with ammonium chloride(Boyle, W. Transplantation 1968.6:71) and when cell selection wasperformed dead cells were removed by the method described by von Boehmerand Shortman (von Boehmer, H. and Shortman, K, J. Immunol, Methods1973:1:273). The cells were suspended in phosphage buffered saline(PBS), 1% Bovine serum albumen, 0.01% sodium azide; throughout all cellselection procedures the cells were kept at 4° C. in this medium.

2.4 Cell Solution

Biotinylated NIP-BSA was coupled to streptavidin coupled magnetic beads(Dynabeads M280 Streptavidin, Dynal, Oslo, Norway) by incubating 10⁸beads with 100 μg of biotinylated protein for 1 hour, with occasionalagitation, and then washing five times to remove unbound antigen. Thecoupled beads were stored at 4° C. in medium until required. Forselection of antigen binding cells the cells (2-4×10⁷/ml) were firstincubated for 30 minutes with uncoupled beads, at a bead:cell ratio of1:1, to examine the degree of non-specific binding. The beads were thenseparated by placing the tube in a magnetic device (MPC-E Dynal) for 3-5minutes. The unbound cells were removed and then incubated with NIP-BSAcoupled magnetic beads, at a bead:cell ratio of 0.1:1, for 60 minutes,with occasional agitation. The beads and rosetted cells were separatedas described above. The beads were then resuspended in 1 ml of mediumand the separation repeated; this process was repeated 5-7 times untilno unbound cells could be detected when counted on a haemocytometer.

For the depletion of surface immunoglobulin positive cells the cellswere incubated with 20 μg biotinylated goat anti-mouse polyvalentimmunoglobulin (Sigma, Poole, UK). The cells were then washed twice withmedium and added to streptavidin coupled magnetic beads at a bead tocell ratio of 30:1. After 30 minutes incubation the beads and rosettedcells were separated by applying the magnetic device three times—takingthe supernatant each time.

2.4 DNA/cDNA Preparation, PCR Amplification and Cloning

DNA was prepared by a simple proteinase-K digest method that wasparticularly convenient for small numbers of cells (PCR Protocols: AGuide to Methods and Applications. Ed Innis M. A., Gelfand D. H.,Sninsky J. J. and White T. J. Academic Press). RNA preparation andsubsequent cDNA synthesis was performed as described by Gherardi et al(Gherardi E., Pannell R. and Milstein C. J. Immunol. Methods, 1990.126:61-68). PCR and cloning of the heavy chain libraries was performedusing the primers and conditions described by Ward et al (Ward, E. S.,Güssow, D., Griffiths, A. D., Jones, P. T. and Winter, G., Nature, 1989.341: 544-546); 40 cycles of PCR amplification were performed. The VH andFv expression vectors used were adapted from those previously describedby Ward et al. They were both subcloned into pUC119 (Veira and Messingsee later) and the Fv expression vector was modified to include agermline lambda-1 light chain (obtained as a gift from T. Simon(originally cloned by Siegfried Weiss, Basel Institute of Immunology)).The vector is shown in FIG. 53.

2.5 Expression and ELISA

For screening single Colonies were picked into individual wells ofmicrotitre plates (Bibby) in 200 μl 2×TY/Ampicillin 100 μg/ml/0.1%glucose and then incubated at 37° C. for 5-6 hours with agitation,Isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma, Poole, UK) was thenadded to a final concentration of 1 mM and the incubation continued fora further 16 hours at 30° C. before harvesting the supernatants. Thewells of Falcon ELISA plates (Becton Dickenson, N.J., USA) were coatedovernight at room temperature with NIP₁₀-BSA (40 μg/ml in PBS) and thenblocked with 2% skimmed milk powder in PBS for 2 hours at roomtemperature. The bacterial supernatants were added and incubated at roomtemperature for 1 hour and then the plates were washed three times withPBS. Peroxidase conjugated-Goat anti-mouse lambda-chain (SouthernBiotechnology, Birmingham, USA) was added and again incubated for 1 hourat room temperature before washing six times with PBS and thendeveloping with 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(Sigma, Poole, UK) as the peroxidase substrate. The optical density at405 nm was measured using a Thermomax microplate reader (MolecularDevices, Menlo Park, USA) after 30 minutes. Western blotting using theC-terminal myc tag as described in example 27.

3.1 Comparison of RNA/DNA and Antigen Selected Cells

The results of antigen selection are shown in Table 13. Less than 1% ofcells bind to NIP-BSA coated beads and the non-specific binding is verylow. Assessment of the proportion of expressed genes from each VHlibrary using western blotting showed that full length VH domains wereexpressed in 95% (19/20) of all clones when RNA was used as the startingmaterial but only 60% (12/20) of clones when DNA (either selected cellsor from total spleen) was used as the starting material. This differenceprobably results from the fact that many re-arranged pseudogenes couldbe amplified with our primers and it appears that there must be somedegree of selection, at the level of transcription, for functionalgenes.

A variable number of clones from each type of library were screened forthe production of Fv fragments that bound to NIP. Initial screeningELISAs were performed and positives taken to include those with anoptical density of at least twice the background. The initial positiveswere retransformed and the binding checked in duplicate; it wasconfirmed that the binding was specific to NIP and not to BSA. Thefrequency of confirmed positive NIP binding clones for each startingmaterial are shown in Table 14. Using DNA as the starting material forthe PCR amplification is approximately equivalent to sampling the cellspresent as there is only one functional re-arranged heavy chain gene andat most one re-arranged pseudogene per B-cell. Amplifying from the RNAof an animal of course biases the repertoire to the reacting B-cells andin a recently immunised animal this would be expected to give some biastowards the immunogen. The data in Table 14 clearly shows how powerfulthis selection is with the number of antigen specific genes beingenriched at least 96 fold when RNA made one week after primaryimmunisation is used as the starting material. The data also show thatselection for antigen binding cells also provides an alternativepowerful method of selection for the required genetic starting material.

3.2 Comparison of Total Spleen/Surface Immunoglobulin Depleted Spleen

To examine the cellular basis of the selection achieved by using RNA asthe starting material we depleted the spleen of surface immunoglobulinpositive cells using biotinylated anti-polyvalent immunoglobulin andstreptavidin conjugated magnetic beads. Prior FACS analysis haddemonstrated that this method removed over 96% of surface immunoglobulinpositive cells. RNA was prepared from both surface immunoglobulindepleted and non-depleted factions of a spleen and VH libraries madefrom each. The ELISA results (Table 14) show that the number ofpositives is certainly not decreased by this depletion suggesting thatthe major portion of the selective effect of using RNA may come fromsurface immunoglobulin negative G-cells (probably plasma cells).

CONCLUSIONS

The applicants have demonstrated the importance of the amplification ofspecific RNA produced by immunisation to enable binding activity to beobtained with any reasonable frequency from a combinatorial library. Theapplicants have also demonstrated an alternative strategy which mimicsthat of the immune system itself. Using a simple method of selecting forantigen binding cells gave comparable enrichment and has the addedadvantage of using a broader range of genes. At first sight the randomcombinatorial approach would appear unlikely to produce the originalcombination of heavy and light chain because of the vast diversity ofthe immunoglobulin genes. The applicants show here, however, thatfollowing immunisation, with a good antigen, 10% of the VH genes fromtotal splenic RNA isolated come from antigen specific cells so theeffective size of the repertoire is greatly reduced. This together withthe fact that promiscuity of the heavy and light chains occurs (examples21 and 22) accounts for the fact that combinatorial system does produceantigen binding clones with reasonable frequency. The data also suggeststhat the bulk of the antigen specific RNA comes from surfaceimmunoglobulin negative cells which are most likely plasma cells.

The data also show that this simple method of antigen selection may beuseful in reducing the complexity of the combinatorial library. In thiscase an enrichment of antigen specific genes of at least 56 fold hasbeen achieved which in the normal case where heavy and light chains areunknown would result in a reduction of the complexity of thecombinatorial library by a factor of over 3000. A further advantage ofusing antigen selected cells (and amplifying from DNA to reduce any biasdue to the state of the cell) is that this results in a broader range ofantibody genes amplified. It may be that a simple cell selection such asthat the applicants have described here in combination with phageselection would be ideal. From this example it can be seen that bycombining cell and phage selection methods one could reasonably expectto screen all the combinations of heavy and light chain (approximately4×10¹⁰) and would thus be able to screen all binding combinationsalthough this would not, at present, be possible from whole spleen(approximately 4×10¹⁴ combinations, assuming 50% B-cells).

TABLE 1 Enrichment of pAb (D1.3) from vector population INPUT OUTPUTRATIO RATIO^(a) oligo^(b) ELISA^(c) pAb:fd-CAT1 pAb:total phagepAb:total phage ENRICHMENT^(d) Single Round 1:4 × 10³ 43/124 1.3 × 10³1:4 × 10⁴ 2/82 1.0 × 10³ Two Rounds 1:4 × 10⁴ 197/372  2.1 × 10⁴ 1:4 ×10⁵ 90/356 3/24 1.0 × 10⁵ 1:4 × 10⁶ 27/183 5/26 5.9 × 10⁵ 1:4 × 10⁷13/278 1.8 × 10⁶ Footnotes: ^(a)Approximately 10¹² phage with the statedratio of pAb (D1.3):FDTPs/Bs were applied to 1 ml lysozyme-sepharosecolumns, washed and eluted. ^(b)TG1 cells were infected with the elutedspecific binding phage and plated onto TY-tet plates. After overnightincubation at 30-37° C., the plates were analysed by hydridisation tothe ³²p, labelled oligonucleotide VH1FOR (Ward et al op cit) which isspecific to pAb D1.3. ^(c)Single colonies from overnight plates weregrown, phage purified, and tested for lysozyme binding. ^(d)Enrichmentwas calculated from the oligonucleotide probing data.

TABLE 2 Enrichment of pAb (D1.3) from mixed pAb population Input Ratio¹Output Ratio² (pAbD1.3:pAbNQ11) (pAb D1.3:Total phage) Enrichment SingleRound 1:2.5 × 10⁴  18/460 0.98 × 10³ 1:2.5 × 10⁵  3/770 0.97 × 10³ 1:2.5× 10⁴  0/112 — pAb NQ11 only  0/460 — Second Round 1:2.5 × 10⁴ 119/1701.75 × 10⁴ 1:2.5 × 10⁵ 101/130 1.95 × 10⁵ 1:2.5 × 10⁶ 102/204 1.26 × 10⁶1:2.5 × 10⁷  0/274 — 1:2.5 × 10⁸  0/209 — pAb NQ11 only  0/170 — Notes¹10¹⁰ phage applied to a lysozyme column as in table 1. ²Plating ofcells and probing with oligonucleotide as in table 1, except theoligonucleotide was D1.3CDR3A.

TABLE 3 Enzyme activity of phage-enzyme No. of molecules ng of enzymeRate of Enzyme equivalent Input or No. of phage (OD/hr) (×10⁻¹¹) PureEnzyme 335 34 24.5 Pure Enzyme 177.5 17.4 12.25 Pure Enzyme 88.7 8.76.125 Pure Enzyme 44.4 4.12 3.06 Pure Enzyme 22.2 1.8 1.5 Pure Enzyme11.1 0.86 0.76 No Enzyme 0 0.005 0 fd-phoAla166/TG1 1.83 × 10¹¹ 5.82 4.2fd-CAT2/TG1  1.0 × 10¹² 0.155 0.112 fd-phoAla166/KS272  7.1 × 10¹⁰ 10.327.35 fd-CAT2/KS272  8.2 × 10¹² 0.038 0.027

TABLE 4 Affinity selection of hapten-binding phage. Clones binding tophOx^(†) Pre-column After first round After second round After thirdround A Random Combinatorial Libraries phOx-immunised mice  0/568 (0%)48/376 (13%)   175/188 (93%) Unimmunised mice  0/388 (0%) B HierarchicalLibraries VH-B/Vκ-rep library  6/190 (3%) 348/380 (92%)   VH-rep/Vκ-dlibrary  0/190 (0%) 23/380 (7%)   C Fractionation of VII-B/Vκ-d andVII-B/Vκ-b phage^(†) Mixture of clones  88/1896 (4.6%)  55/95 (57.9%) 1152/1156 (99.7%) 1296/1299 (99.8%)   [44/1740 (2.5%)*] ^(†)In panel C,numbers refer to VH-B/Vκ-d colonies. Numbers after three reinfectionsand cycles of growth. This control, omitting the column steps, confirmsthat a spurious growth or infectivity advantage was not responsible forthe enrichment for clone VH-B/Vκ-d.

TABLE 5 Binding to Chain(s) Chain as Soluble Phage/Phagemid^(†) HelperPhage phOx* displayed^(#) gene III fusion^(#) chain(s)^(#) A fd CAT2 nonbinding none fd CAT2-I binding scFv scFv fd CAT2-II binding Fab lightchain heavy chain pHEN1 VCSMB non binding none pHEN1 I VCSMB bindingscFv scFv pHEN1 II VCSMB binding Fab light chain heavy chain B pHEN1 I(HB2151) binding scFv^(§) pHEN1 II (HB2151) binding Fab^(§) C fdCAT2-III non binding heavy chain heavy chain fd CAT2-IV non bindinglight chain light chain pHEN1 III (HB2151) VCSMB non binding none heavychain pHEN1 III (HB2151) fd-tet-DOG1 IV binding Fab light chain heavychain pHEN1 IV (HB2151) VCSMB non binding none light chain pHEN1 IV(HB2151) fd-tet-DOG1 III binding Fab heavy chain light chain Overview ofphOx-BSA ELISA results of phage and phagemid constructions. *Phase wereconsidered to be ‘binding’ if OD₄₀₅ of sample was at least 10 foldgreater than background in ELISA; ^(†) E. coli TG1 was used for thegrowth of the phage unless the use of E.coli HB2151 is specificallyindicated; ^(#)Information deduced from genetic structure and inaccordance with binding data; ^(§)Result confirmed experimentally byWestern blot (for Fab, see FIG. 29.

TABLE 6 Kinetic parameters of soluble and phage-bound alkalinephosphase. Relative values of k_(cat) and K_(m) for the soluble enzymeand for the phage enzyme were derived by comparing with the values forwild type enzyme (phoArg166) and the phage-wild type enzyme(fdphoArg166). Soluble enzyme (Data from Phage enzyme Chaidaroglu et al1988) (Data from this study) phoArg166 phoAla166 phoArg166 phoAla166K_(m) (μM) 12.7 1620 73 1070 Relative K_(m) 1 127 1 14.6 Relative 10.397 1 0.360 k_(cat) Relative 1 0.0032 1 0.024 k_(cat)/K_(m)

TABLE 7 Enzyme Activity of Phage Samples SPECIFIC ACTIVITY INPUT PHAGERATE (mol substrate SAMPLE PARTICLE (pmol substrate converted/mol(Construct:host) (pmol converted/min) phage/min) fdphoArg166: 2.3 86953700 TG1 fdphoAla166: 5.6 2111 380 TG1 fdphoAla166: 1.8 2505 1400 KS272fdCAT2: 3.3 <1 <0.3 TG1 fdCAT2: 5.6 70 12 KS272

TABLE 8 Affinity chromatography of phage-enzymes INFECTIVITY (Percentageof phage particles INPUT PHAGE OUTPUT PHAGE which are PARTICLE PARTICLESAMPLE infectious) (×10⁹) (×10⁹) fdphoArg166 0.37% 5160 30 fdphoAla1660.26% 3040 90 fdCAT2 4.75% 4000 2

TABLE 9 Mutations in scFvB18 selected by display on phage followinggrowth in mutator strains Nucleotide mutation (base position) Amino acidmutation Number 308 Ala -> Val (VH FR3) 3 703 Tyr -> Asp (VL CDR3) 1 706Ser -> Gly (VL CDR3) 1 724 Gly -> Ser (VL FR4) 21 725 Gly -> Asp (VLFR4) 3 734 Thr -> Ile (VL FR4) 1

TABLE 10 (i) Oligonucleotide primers used for PCR of humanimmunoglobulin genes Oligo Name Sequence Human VH Back PrimersHuVH1aBACK 5′-CAG GTC CAG CTG GTG CAG TCT GG-3′ (SEQ ID NO: 81)HuVH2aBACK 5′-CAG GTC AAC TTA AGG GAG TCT GG-3′ (SEQ ID NO: 82)HuVH3aBACK 5′-GAG GTG CAG CTG GTG GAG TCT GG-3′ (SEQ ID NO: 83)HuVH4aBACK 5′-CAG GTG CAG CTG CAG GAG TCG GG-3′ (SEQ ID NO: 84)HuVH5aBACK 5′-GAG GTG CAG CTG TTG CAG TCT GG-3′ (SEQ ID NO: 85)HuVH6aBACK 5′-CAG GTA CAG CTG CAG CAG TCA GG-3′ (SEQ ID NO: 86)HuVH1aBACKSfi 5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAGCTG GTG CAG TCT GG-3′ (SEQ ID NO: 87) HuVH2aBACKSfi 5′-GTC CTC GCA ACTGCG GCC CAG CCG GCC ATG GCC CAG GTC AAC TTA AGG GAG TCT GG-3′ (SEQ IDNO: 88) HuVH3aBACKSfi 5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAGGTG CAG CTG GTG GAG TCT GG-3′ (SEQ ID NO: 89) HuVH4aBACKSfi 5′-GTC CTCGCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG CAG GAG TCG GG-3′(SEQ ID NO: 90) HuVH5aBACKSfi 5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATGGCC CAG GTG CAG CTG TTG CAG TCT GC-3′ (SEQ ID NO: 91) HuVH6aBACKSfi5′-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTA CAG CTG CAG CAGTCA GG-3′ (SEQ ID NO: 92) Human JH Forward Primers HuJH1-2FOR 5′-TGA GGAGAC GGT GAC CAG GGT GCC-3′ (SEQ ID NO: 93) HuJH3FOR 5′-TGA AGA GAC GGTGAC CAT TGT CCC-3′ (SEQ ID NO: 94) HuJH4-5FOR 5′-TGA GGA GAC GGT GAC CAGGGT TCC-3′ (SEQ ID NO: 95) HuJH6FOR 5′-TGA GGA GAC GGT GAC CGT GGTCCC-3′ (SEQ ID NO: 96) Human Heavy Chain Constant Region PrimersHuIgG1-4CH1FOR 5′-GTC CAC CTT GGT GTT GCT GGG CTT-3′ (SEQ ID NO: 97)HuIgMFOR 5′-TGG AAG AGG CAC GTT CTT TTC TTT-3′ (SEQ ID NO: 98) Human VκBack Primers HuVκ1aBACK 5′-GAC ATC CAG ATG ACC CAG TCT CC-3′ (SEQ ID NO:99) HuVκ2aBACK 5′-GAT GTT GTG ATG ACT CAG TCT CC-3′ (SEQ ID NO: 100)HuVκ3aBACK 5′-GAA ATT GTG TTG ACG CAG TCT CC-3′ (SEQ ID NO: 101)HuVκ4aBACK 5′-GAC ATC GTG ATG ACC CAG TCT CC-3′ (SEQ ID NO: 102)HuVκ5aBACK 5′-GAA ACG ACA CTC ACG CAG TCT CC-3′ (SEQ ID NO: 103)HuVκ6aBACK 5′-GAA ATI GTG CTG ACT CAG TCT CC-3′ (SEQ ID NO: 104) HumanJκ Forward Primers HuJκ1FOR 5′-ACG TTT GAT TTC CAC CTT GGT CCC-3′ (SEQID NO: 105) HuJκ2FOR 5′-ACG TTT GAT CTC CAG CTT GGT CCC-3′ (SEQ ID NO:106) HuJκ3FOR 5′-ACG TTT GAT ATC CAC TTT GGT CCC-3′ (SEQ ID NO: 107)HuJκ4FOR 5′-ACG TTT GAT CTC CAC CTT GGT CCC-3′ (SEQ ID NO: 108) HuJκ5FOR5′-ACG TTT AAT CTC CAG TCG TGT CCC-3′ (SEQ ID NO: 109) HuJκ1BACKNot5′-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT TTC CAC CTT GGT CCC-3′(SEQ ID NO: 110) HuJκ2BACKNot 5′-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTTGAT CTC CAG CTT GGT CCC-3′ (SEQ ID NO: 111) HuJκ3BACKNot 5′-GAG TCA TTCTCG ACT TGC GGC CGC ACG TTT GAT ATC CAC TTT GGT CCC-3′ (SEQ ID NO: 112)HuJκ4BACKNot 5′-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAC CTTGGT CCC-3′ (SEQ ID NO: 113) HuJκ5BACKNot 5′-GAG TCA TTC TCG ACT TGC GGCCGC ACG TTT AAT CTC CAG TCG TGT CCC-3′ (SEQ ID NO: 114) Human κ ConstantRegion Primers HuCκFOR 5′-AGA CTC TCC CCT GTT GAA GCT CTT-3′ (SEQ ID NO:115) HuCκFORNotl 5′-GAG TCA TTC TCG ACT TGC GGC CGC TTA TTA AGA CTC TCCCCT GTT GAA GCT CTT-3′ (SEQ ID NO: 116) HuCκFORNot2 5′-GAG TCA TTC TCGACT TGC GGC CGC AGA CTC TCC CCT GTT GAA GCT CTT-3′ (SEQ ID NO: 117)Human λ Back Primers HuVλ1BACK 5′-CAG TCT GTG TTG ACG CAG CCG CC-3′ (SEQID NO: 118) HuVλ2BACK 5′-CAG TCT GCC CTG ACT CAG CCT GC-3′ (SEQ ID NO:119) HuVλ3aBACK 5′-TCC TAT GTG CTG ACT CAG CCA CC-3′ (SEQ ID NO: 120)HuVλ3bBACK 5′-TCT TCT GAG CTG ACT CAG GAC CC-3′ (SEQ ID NO: 121)HuVλ4BACK 5′-CAC GTT ATA CTG ACT CAA CCG CC-3′ (SEQ ID NO: 122)HuVλ5BACK 5′-CAG GCT GTG CTC ACT CAG CCG TC-3′ (SEQ ID NO: 123)HuVλ6BACK 5′-AAT TTT ATG CTG ACT CAG CCC CA-3′ (SEQ ID NO: 124) Human λForward Primers HuJλ1FOR 5′-ACC TAG GAC GGT GAC CTT GGT CCC-3′ (SEQ IDNO: 125) HuJλ2-3FOR 5′-ACC TAG GAC GGT CAG CTT GGT CCC-3′ (SEQ ID NO:126) HuJλ4-5FOR 5′-ACC TAA AAC GGT GAG CTG GGT CCC-3′ (SEQ ID NO: 127)HuJλFORNOT 5′-GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT GAC CTTGGT CCC-3′ (SEQ ID NO: 128) HuJλ2-3FORNOT 5′-GAG TCA TTC TCG ACT TGC GGCCGC ACC TAG GAC GGT CAG CTT GGT CCC-3′ (SEQ ID NO: 129) HuJλ4-5FORNOT5′-GAG TCA TTC TCG ACT TGG GGC CGC ACY TAA AAC GGT GAG CTG GGT CCC-3′(SEQ ID NO: 130) Human λ Constant Region Primers HuCλFOR 5′-TGA AGA TTCTGT AGG GGC CAC TGT CTT-3′ (SEQ ID NO: 131) HuCλFORNot1 5′-GAG TCA TTCTCG ACT TGC GGC CGC TTA TTA TGA AGA TTC TGT AGG GGC CAC TGT CTT-3′ (SEQID NO: 132) HuCλFORNot2 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGC AGA TTCTGT AGG GGC TGT CTT-3′ (SEQ ID NO: 133) Linker oligos Reverse JH forscFv linker RHuJH1-2 5′-GCA CCC TGG TCA CCG TCT CCT CAG GTG G-3′ (SEQ IDNO: 134) RHuJH3 5′-GGA CAA TGG TCA CCG TCT CTT CAG GTG G-3′ (SEQ ID NO:135) RHuJH4-5 5′-GAA CCC TGG TCA CCG TCT CCT CAG GTG G-3′ (SEQ ID NO:136) RHuJH6 5′-GGA CCA CGG TCA CCG TCT CCT CAG GTG C-3′ (SEQ ID NO: 137)Reverse IgG1-4CH1 primer for Fab linker RhuIgGl-4CH1FOR 5′-AAG CCC AGCAAC ACC AAG GTG GAC-3′ (SEQ ID NO: 138) Reverse Vκ for scFv linkerRhuVκ1aBACKFv 5′-GGA GAC TGG GTC ATC TGG ATG TCC GAT CCG CC-3′ (SEQ IDNO: 139) RhuVκ2aBACKFv 5′-GGA GAC TGA GTC ATC ACA ACA TCC GAT CCG CC-3′(SEQ ID NO: 140) RhuVκ3aBACKFv 5′-GGA GAC TGC GTC AAC ACA ATT TCC GATCCG CC-3′ (SEQ ID NO: 141) RhuVκ4aBACKFv 5′-GGA GAC TGG GTC ATC ACG ATGTCC GAT CCG CC-3′ (SEQ ID NO: 142) RhuVκ5aBACKFv 5′-GGA GAC TGC GTG AGTGTC GTT TCC GAT CCG CC-3′ (SEQ ID NO: 143) RhuVκ6aBACKFv 5′-GGA GAC TGAGTC AGC ACA ATT TCC GAT CCG CC-3′ (SEQ ID NO: 144) Reverse Vκ for Fablinker RHuVκ1aBACKFab 5′-GGA GAC TGG GTC ATC TGG ATG TCG GCC ATC GCTGG-3′ (SEQ ID NO: 145) RHuVκ2aBACKFab 5′-GGA GAC TGC GTC ATC ACA ACA TCGGCC ATC GCT GG-3′ (SEQ ID NO: 146) RHuVκ3aBACKFab 5′-GGA GAC TGC GTC AACACA ATT TCG GCC ATC GCT GG-3′ (SEQ ID NO: 147) RHuVκ4aBACKFab 5′-GGA GACTGG GTC ATC ACG ATG TCG GCC ATC GCT GG-3′ (SEQ ID NO: 148)RHuVκ5aBACKFab 5′-GGA GAC TGC GTG AGT GTC GTT TCG GCC ATC GCT GG-3′ (SEQID NO: 149) RHuVκ6aBACKFab 5′-GGA GAC TGC GTC AGC ACA ATT TCG GCC ATCGCT GG-3′ (SEQ ID NO: 150) Reverse Vλ for svFv linker RHuVλBACK1Fv5′-GGC GGC TGC GTC AAC ACA GAC TGC GAT CCG CCA CCG CCA GAG-3′ (SEQ IDNO: 151) RHuVλBACK2Fv 5′-GCA GGC TGA GTC AGA GCA GAC TGC GAT CCG CCA CCGCCA GAG-3′ (SEQ ID NO: 152) RHuVλBACK3aFv 5′-GGT GGC TGA GTC AGC ACA TAGGAC GAT CCG CCA CCG CCA GAG-3′ (SEQ ID NO: 153) RHuVλBACK3bFv 5′-GGG TCCTGA GTC AGC TCA GAA GAC GAT CCG CCA CCG CCA GAG-3′ (SEQ ID NO: 154)RHuVλBACK4Fv 5′-GGC GGT TGA GTC AGT ATA ACG TGC GAT CCG CCA CCG CCAGAG-3′ (SEQ ID NO: 155) RHuVλBACK5Fv 5′-GAC GGC TGA GTC AGC ACA GAC TGCGAT CCG CCA CCG CCA GAG-3′ (SEQ ID NO: 156) RHuVλBACK6Fv 5′-TGG GGC TGAGTC AGC ATA AAA TTC GAT CCG CCA CCG CCA GAG-3′ (SEQ ID NO: 157) ReverseVλ for Fab linker RHuVλBACK1Fab 5′-GGC GGC TGC GTC AAC ACA GAC TGG GCCATC GCT GGT TGG GCA-3′ (SEQ ID NO: 158) RHuVλBACK2Fab 5′-GCA GGC TGA GTCAGA GCA GAC TGG GCC ATC GCT GGT TGG GCA-3′ (SEQ ID NO: 159)RHuVλBACK3aFab 5′-GGT GGC TGA GTC AGC ACA TAG GAG GCC ATC GCT GGT TGGGCA-3′ (SEQ ID NO: 160) RHuVλBACK3bFab 5′-GGG TCC TGA GTC AGC TCA GAAGAG GCC ATC GCT GGT TGG GCA-3′ (SEQ ID NO: 161) RHuVλBACK4Fab 5′-GGC GGTTGA GTC AGT ATA ACG TGG GCC ATC GCT GGT TGG GCA-3′ (SEQ ID NO: 162)RHuVλBACK5Fab 5′-GAC GGC TGA GTC AGC ACA GAC TGG GCC ATC GCT GGT TGGGCA-3′ (SEQ ID NO: 163) RHuVλBACK6Fab 5′-TGG GGC TGA GTC AGC ATA AAA TTGGCC ATC GCT GGT TGG GCA-3′ (SEQ ID NO: 164)

TABLE 11 Deduced protein sequences of heavy and light chains selectedfrom unimmunized library Oxazolone binder HEAVY CHAIN VH15.4QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYGIS WVRQAPGQGLEWMG WISAYNGNTKYAQKLQGRVTMITDTSTSTAYMELRSLRSDDTAVYYCVR LLPKRTATLH YYIDVWGKGT (SEQ ID NO: 165)LIGHT CHAIN VL15.4 NNYVS WYQHLPGTAPNLLIY DNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYC GIWDGR (SEQ ID NO: 166) BSA BindersHEAVY CHAINS VH3.5 QVQLVQSGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVAVISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK TGYSSGWGYFDYWGQGT(SEQ ID NO: 167) LIGHT CHAINS VL3.5 SSELTQDPAVSVALGQTVRITCQGDSLRSYYAS WYQQKPGQAPVLVIY GKNNRPS GIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNH VVFGG (SEQ ID NO: 168) Lysozyme binders: HEAVY CHAINS VH10.1SLTCSVSGDSIS SGGYS WIRQPSGKGLEWIG SVHHSGPTYYNPSLKSRVTMSVDTSKNQFSLKLKSVTAADTAMYFCAR EGGSTWRSLYKH YYMDVWGK (SEQ ID NO: 169)VH14.1 QVQLQESGPGLVKPSETLSLVCTVSGGSLS FSYWG WIRQPPGKGLEWIGYISHRGTDYNSSLQS RVTISADTSKNQFSLKLSSVTAADTAVYYCAR SFSNSFFFGY WGQGT (SEQID NO: 170) VH13.1 QVQLVQSGAEVKKPGQSLMISCQGSGYSFS NYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQG QVTISADKSISTAYLHWSSLKASDTALYYCAR LVGGTPAY WGQGT (SEQID NO: 171) VH16.1 QVQLVQSGAEVKKPGQSLRISCKGAGYSFS TYWIG WVRQMPGKGLEWMGIIYPDDSDTRYSPSFEG QVTISVDKSITTAYLHWSSLKA (SEQ ID NO: 172) LIGHT CHAINSVK10.1 EIVLTQSPSSLSASVGDRVTTTC RASQSISNYLN WYQQKPGKAPKLLIY AASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQTIISFP LTFGGG (SEQ ID NO: 173) VL14.1SSELTQDPAVSVAFGQTVRITC QGDSLRSSYAS WYQQKPGQAPLLVIY GENSRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC NSRDSRGTHL EVFGG (SEQ ID NO: 174)VL13.1 HVILTQPASVSGSPGQSITISC TGSSRDVGGYNYVS WYQHHPGKAPKLLIS EVTNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYFC ASYTSSKT YVFGG (SEQ ID NO: 175) VL16.1QSALTQPASVSGSPGQSITISC SGSSSDIGRYDYVS WYQHYPDKAPKLLIY EVKHRPSGISHRFSASKSGNTASLTISELQPGDEADYYC ASYT(SEQ ID NO: 176)

TABLE 12 Enrichment of pAbNQ11 from pAbD1.3 background by affinityselection using Ox-BSA biotinylated with a cleavable reagent and bindingto streptavidin magnetic beads Input Ratio¹ Output Ratio²(pAbD1.3:pAbNQ11) (pAb NQ11:Total phage) Enrichment  2235:1 61/197 69022350:1  5/202 544 ¹1.9 × 10¹⁰ phage in 0.5 ml mixed for 1 hour with 5μl streptavidin-magnetic beads precoated with antigen (OX-BSA).²Colonies probed with the oligonucleotide NQ11CDR3

TABLE 13 Results of antigenic cell selection Number % of total of Cellscells Total spleen cells   4 × 10⁷ Cells bound to 0.8 × 10⁴ 0.02uncoated beads Cells bound to NIP-BSA  22 × 10⁴ 0.55 coated beads

TABLE 14 Results of Fv NIP binding ELISAs from selected cellpopulations: *Degree of Positives Enrichment Cell Population DNA fromtotal spleen  0/940 — RNA from total Spleen 29/282 >96 DNA from antigen17/282 >56 binding cells Surface Ig Selection RNA fromSurface Ig 8/94 —negative fraction RNA from total Spleen 4/94 — *Degree of enrichmentcompared to total DNA.

1. Recombinant host cells each of which harbors a phagemid comprising anucleic acid fragment encoding one member of a specific binding pairfused to a nucleic acid encoding a gene III coat protein surfacecomponent of a filamentous bacteriophage and further comprising anorigin of replication of a filamentous bacteriophage, the gene III coatprotein surface component encoding nucleic acid and the origin ofreplication being the only nucleic acid in the phagemid derived fromfilamentous bacteriophage, whereby the host cells collectively harbor inthe phagemids a library of nucleic acid fragments encoding a geneticallydiverse population of the specific binding pair members, each member ofthe specific binding pair capable of being expressed as a fusion proteinwith the gene III coat protein surface component of a filamentousbacteriophage so that each member of the specific binding pair comprisesa functional specific binding domain for its complementary specificbinding pair member and whereby upon infection of said recombinant hostcells with a helper phage, the phagemids are each packaged intofilamentous bacteriophage particles displaying on their surface thefunctional specific binding pair member as a fusion with the gene IIIsurface component of the filamentous bacteriophage and whereby eachfilamentous bacteriophage has a coat partially derived from the helperphage and partly from said fusion.
 2. The recombinant host cellsaccording to claim 1 wherein said genetically diverse population isderived from in vitro mutagenesis of nucleic acid encoding a member of aspecific binding pair.
 3. The recombinant host cells according to claim2 wherein said member of a specific binding pair comprises a bindingdomain of an immunoglobulin.
 4. The recombinant host cells according toclaim 1 wherein said genetically diverse population is derived from arepertoire of rearranged immunoglobulin genes of an animal immunizedwith complementary specific binding pair member.
 5. The recombinant hostcells according to claim 1 wherein said genetically diverse populationis derived from a repertoire of rearranged immunoglobulin genes of ananimal not immunized with complementary specific binding pair member. 6.The recombinant host cells according to claim 3 wherein said member of aspecific binding pair is a scFv molecule.
 7. The recombinant host cellsaccording to claim 1 wherein said member of a specific binding pair is ascFv molecule.
 8. The recombinant host cells according to claim 4wherein said member of a specific binding pair is a scFv molecule. 9.The recombinant host cells according to claim 5 wherein said member of aspecific binding pair is a scFv molecule.